Business and data processing system for providing mass spectrometric services

ABSTRACT

Embodiments of business systems and data processing systems and related methods, apparatus, compositions, systems, and articles of manufacture useful for providing mass spectrographic analysis services are disclosed. In embodiments, MS analysis services may be provided to customers without exposing proprietary information of the customer to the service provider or others.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. non-provisionalapplication Ser. No. 14/628,287, filed Feb. 22, 2015, entitled “Methods,apparatus, and compositions for mass spectrometry,” issuing as U.S. Pat.No. 9,460,904 on Oct. 4, 2016, which is a continuation-in-part of U.S.non-provisional application Ser. No. 14/153,078, filed Jan. 13, 2014,entitled “Mass spectrometry methods and apparatus”, issued as U.S. Pat.No. 9,082,600 on Jul. 14, 2015, which claims priority from U.S.Provisional Patent Application No. 61/751,078, of the same title, filedJan. 10, 2013, and of U.S. non-provisional application Ser. No.13/374,909, entitled “Substrate compositions and methods of usethereof”, filed Jan. 23, 2012, issuing as U.S. Pat. No. 8,963,080 onFeb. 24, 2015, which claims priority from U.S. provisional applicationNo. 61/461,690, filed Jan. 22, 2011, of the same title; this applicationclaims the benefit of and priority from all of the foregoing, thecontents of which are incorporated herein by reference as though setforth in full.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

None

TECHNICAL FIELD

The present disclosure relates to the field of mass spectrometry.

BACKGROUND

A wide range of pharmaceutical, scientific, chemical, consumer,industrial, clinical and diagnostic markets depend on the utilization oflarge libraries of samples. For example, small molecule compoundlibraries and screens are at the heart of the pharmaceutical industry.These libraries often contain many hundreds of thousands of smallmolecule samples, typically dissolved in DMSO or enzyme assay buffer, inmultiwell plates that occupy many cubic feet of space and often requirecontrolled environments. The scale of these libraries makes theircharacterization or use in assays difficult using conventionalapproaches. Even simple assays measuring library integrity, stabilityand solubility are extremely difficult and often not performed due tothe limited throughput of chemically specific analytical assays used bythe group possessing the library. Therefore, it would be desirable toprovide this library to a third party for analysis. However, the valueof the library itself and often the intellectual property related to thelibrary makes shipping chemical libraries and screens off-siteundesirable. In addition, enzymatic assay screens using the compoundlibrary along with additional assay-specific components are highlyvaluable not only due to the composition of the assay but also becauseof the proprietary information that they contain. This inability tocharacterize libraries limits industrial efforts in drug and enzymedevelopment due to lack of information on the library composition and onthe stability and solubility of compounds in standard assay conditions.This may result, for example, in false negatives due to compounds notbeing soluble under test conditions or having degraded in storage.Analytical throughput also limits the experimental space that can beexplored, again missing promising leads and slowing the pace of drug andenzyme development. These considerations make it imperative to developtechnologies that enable off-site analysis, including by third parties,in a format that does not require amounts of sample large enough thatthe proprietary details of the assay could be reverse-engineered, and ina format that is readily transportable while maintaining the integrityof the sample library.

SUMMARY

Large sample libraries are inherently difficult or impossible totransport. Often they occupy room-sized spaces, must be kept under highrefrigeration and tight environmental control, are stored and managedwith massive and complex robotics systems, and contain samples obtainedat great expense and embodying valuable proprietary information. Libraryowners are understandably unwilling to allow valuable samples offpremises and risk accidental destruction, theft, or reverse engineering,and in any case the handling, storage, and transport challenges make itimpracticable to do so in any quantity useful for high throughputscreening.

Recent developments in mass spectrometry (MS) analysis have opened up avast repertoire of new analytical techniques that could contributeentire new dimensions of information for sample library evaluation andscreening. However, these techniques often require analytical expertisepossessed only by a relatively few specialists, and specializedinstrumentation that is expensive to acquire and not readilytransportable.

Disclosed herein are methods, apparatus, compositions, and articles ofmanufacture that, in various embodiments, make possible the transport oflarge sample sets to an offsite location for MS analysis, in aminiaturized array format that minimizes or eliminates the risk ofreverse engineering. The miniaturization is readily accomplished at thelibrary site using easily transportable instrumentation, making itunnecessary for the sample library itself ever to leave its secure,environmentally controlled repository. Only the miniaturized array needleave the library premises, and the miniaturized samples may be trackedand results reported in a blinded fashion whereby no informationregarding the exact chemical composition or provenance of the samples isdivulged, yet the results are provided to those in control of thelibrary in a format that allows them to link the mass spectrometry datato the sample identifiers thereby obtaining information on thecomposition and/or activities of their samples.

Offsite transport of a MS sample array poses novel stability and sampleintegrity problems due to the desire to accommodate standard transportservices such as mail or common carrier, notwithstanding the presence ofsamples of unknown and variable composition and stability, the closespacing of samples to achieve desired miniaturization, the desire toenable the use of MS techniques that may entail relatively weakassociation of samples with the surface and/or the use of potentiallyproblematic coatings or additives, and the risk of contamination andtemperature or atmospheric changes during transport. As disclosed hereinand demonstrated in the examples, the inventors have devised apparatusand methods for stability-packaging the miniaturized library arrays fortransport which is effective to enable long distance transport andmedium term storage while maintaining sample integrity.

The methods and apparatus disclosed herein thus enable a novel businessmodel offering a service to owners of large sample libraries, wherebysamples can be miniaturized onto an MS array at the library site, eitherby the customer or an MS analysis provider. The samples can then betransported to an offsite location where the required instrumentationand expertise is available for a desired MS analysis, and results can bereported back to the customer, while maintaining strict confidentialityof the nature and provenance of the samples.

In general, there are provided methods, devices, apparatus, and articlesof manufacture for the general purpose of characterizing one or moreaspects of the chemical composition or activities of sample librariesincluding, for example, but not limited to, aspects related to thedevelopment of pharmaceuticals, enzyme development, and medicaldiagnostics. These may include, for example, the solubilities of smallmolecules, the stability of small molecules, the reactivity of smallmolecules, composition of biofluids, compositions of tissues,composition of cells, and enzymatic activities.

An object of various aspects of the present disclosure is to provideapparatus, compositions, methods, and articles of manufacture useful forone or more of: miniaturizing large sample libraries onto massspectrometric (MS) arrays, packaging and stabilizing the MS arrays fortransport, tracking the positions of the samples on the array,transporting the MS arrays, performing offsite MS analysis on thearrayed samples, providing customer access to the MS analysis data,linking the mass spectra to library identifiers, and maintaining theconfidentiality of the samples and data throughout the process.

An object of various aspects of the present disclosure is to providehigh density MS arrays whereby hundreds or thousands of samples may beminiaturized onto a single MS substrate.

An object of the present disclosure is to provide a practicable abilityto transport large numbers of samples for offsite analysis by a standardcarrier transport service (i.e. FedEx, USPS, UPS, etc.) withoutexcessive specialized handling during transport that is not standard tothe carrier industry.

An object of various aspects of the present disclosure is to provideapparatus, compositions, methods, and articles of manufacture useful formaintaining integrity of chemical analysis by stabilizing the libraryand preventing or reducing the contamination of MS samples arrayed inhigh density on the MS substrate, whether from foreign substances orfrom cross-contamination between spots or otherwise.

An object of various aspects of the present disclosure is to providecustomers having large sample libraries with practical andcost-effective access to MS analysis requiring specializedinstrumentation and/or expertise.

An object of various aspects of the present disclosure is to facilitatetransport of large sample libraries or portions thereof for offsite MSanalysis in a manner that both preserves sample integrity and allowssamples to leave the library or customer site only in a form providingreasonable assurance against reverse engineering or other unauthorizeduses.

An object of various aspects of the present disclosure is to facilitateoffsite transport of samples that may be arrayed with small separationbetween samples, have unknown and/or widely varying properties, orotherwise be susceptible to a risk of instability or contamination.

An object of various aspects of the present disclosure is to provideconvenient and cost-effective customer access to offsite MS analysisdata that may be queried using blinded tracking identifiers and/orreadily incorporated into existing customer datasets.

Also disclosed herein are various aspects of a desorption ionizationmass spectrometry platform technology that is capable ofhigh-throughput, high-content, label-free mass-based analysis ofmicrovolume quantities of sample. One feature of this technology is thatit capable of rapidly detecting and analyzing thousands of compoundspresent in a complex mixture and is available as a powerful tool for thestudy of biological systems.

It will be apparent to persons of skill in the art that various of theforegoing aspects and/or objects, and various other aspects and/orobjects disclosed herein, can be incorporated and/or achieved separatelyor combined in a single device, method, system, composition, article ofmanufacture, and/or improvement thereof, thus obtaining the benefit ofmore than one aspect and/or object, and that an embodiment may encompassnone, one, or more than one but less than all of the aspects, objects,or features enumerated in the foregoing summary or otherwise disclosedherein. The disclosure hereof extends to all such combinations. Inaddition to the illustrative aspects, embodiments, objects, and featuresdescribed above, further aspects, embodiments, objects, and featureswill become apparent by reference to the drawing figures and detaileddescription. Also disclosed herein are various embodiments of relatedmethods, devices, apparatus, compositions, systems, articles ofmanufacture, and/or improvements thereof. The foregoing summary isintended to provide a brief introduction to the subject matter of thisdisclosure and does not in any way limit or circumscribe the scope ofthe invention(s) disclosed herein, which scope is defined by the claimscurrently appended or as they may be amended, and as interpreted by askilled artisan in the light of the entire disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic depiction in cross-section of an embodiment of astability-packaged MS array.

FIG. 2 is a schematic depiction of an embodiment of a MS substratemounted in a frame.

FIG. 3 shows an embodiment of an exemplary offsite MS analysis systemconsistent with the methods, systems, and apparatus of the presentdisclosure.

FIG. 4 depicts an embodiment of an offsite MS analysis process.

FIG. 5 depicts an embodiment of an MS substrate and a frame including afiducial mark.

FIG. 6 depicts an embodiment of an MS array including MS samples appliedat trackable loci.

FIG. 7 shows an optical microscopic image of an embodiment of anacoustically printed MS array.

FIG. 8 shows an enzyme assay mass spectrum acquired from a trackableposition on an embodiment of an MS array following stability-packagingand transport consistent with the disclosure hereof.

FIG. 9 shows a MS array having a urine sample applied at a trackablelocus in an embodiment consistent with the disclosure hereof.

FIG. 10 shows a mass spectrum obtained from a urine sample applied to atrackable locus on an MS array following stability-packaging andtransport in an embodiment consistent with the disclosure hereof.

FIG. 11 shows a mass spectrum obtained from a drug toxicity assay sampleapplied to a trackable locus on an MS array followingstability-packaging and transport in an embodiment consistent with thedisclosure hereof.

FIG. 12 shows a mass spectrum obtained from a drug molecule sampleapplied to a trackable locus on an MS array followingstability-packaging and transport in an embodiment consistent with thedisclosure hereof.

FIG. 13 depicts schematically the arraying of MS samples onto an MSsubstrate by acoustic deposition in an embodiment consistent with thedisclosure hereof.

FIG. 14 depicts an embodiment of a data management system consistentwith the disclosure hereof.

FIG. 15A shows a mass spectrum of N-Desethylamodiaquine obtained priorto stability packaging and offsite shipping.

FIG. 15B shows a mass spectrum of N-Desethylamodiaquine obtained afterstability packaging and offsite shipping.

FIG. 16 shows a mass spectrum of sample including a blinded analyte withinternal standards obtained after stability packaging and offsitetransport.

FIG. 17 is a spectral comparison of blood sample detected on a substratewith (bottom spectra) and without (top spectra) photoacid treatment ofthe substrate showing enhancement of the ionization peaks detected withthe photoacid treatment.

FIG. 18 shows tissue imaging of a mouse brain slice with and withoutphotoacid treatment, showing significant enhancement of signal withphotoacid treatment (top portion of sample) compared to no treatment(bottom portion of sample).

FIG. 19 shows the intensity map of the spectral data for each compoundin a library analyzed at 30-fold dilution and example spectra that aregenerated for each compound screened.

FIG. 20 shows the spectra obtained from a Raji cell sample treated withrapamycin overlaid with spectra obtained from an untreated sample.

FIG. 21 shows the metabolic profile spectrum of a PNPLA3-knockout mouseliver extract overlaid with the metabolic profile spectrum from the wildtype liver extract showing significant metabolic profile differences.

FIG. 22 shows the ionization profile of a zebrafish sample treated witha library compound detected in the 100 to 1,000 m/z range.

FIG. 23 shows an intensity map of a selected metabolite, phosphocoline,detected in the zebrafish whole organism across a compound library.

FIG. 24 shows the cluster analysis of the zebrafish array in which thespectral profiles obtained from the array were clustered using analgorithm described herein, according to an embodiment consistent withthe disclosure hereof. Distinct groups of cellular or metabolic activity(outlying circles) could be distinguished from background or backgroundactivity (two main clusters of circles).

Figures are not to scale unless expressly so labeled, and relativepositions of objects and components are illustrative. Persons of skillin the art will recognize that many other arrangements, configurations,dimensions, and selections of components are possible and consistentwith the disclosure hereof, and are in no way limited to the embodimentsshown in the figures.

DETAILED DESCRIPTION

Disclosed herein are methods, apparatus, compositions, systems, andarticles of manufacture useful for performing offsite mass spectrometric(MS) analysis of samples. In some embodiments, for example, a pluralityof MS samples from a sample library may be arrayed on one or more MSsubstrates at a loading site where access to the sample library isavailable. The resulting arrays may then be packaged for transport inaccordance with the disclosure hereof to protect the MS samples fromdamage or alteration, and transported to an offsite analysis locationfor MS analysis. Confidentiality as to the identity of the MS samplesmay be preserved by mapping the applied MS samples to arbitrary trackingidentifiers, or by using other blinded tracking strategies, so that noconfidential information on the specific composition, confidentialidentifiers, and/or activities of the library need ever leave thecontrol of the library owner.

FIGS. 3 and 4 depict the broad outlines of an exemplary embodiment of asystem and method for performing offsite MS analysis, of particularutility for analyzing large sample sets, employing various aspects ofthe disclosure hereof. At a loading site 205 under the control of acustomer 200, there is present and accessible a sample library 210, suchas, for example, a drug discovery library. An arraying instrument 215 asdisclosed herein is made available at the loading site. Using thearraying instrument and the methods and apparatus disclosed herein, ananalysis library comprising a selection of MS samples obtained from thesample library is miniaturized by applying 300 the MS samples onto an MSsubstrate as shown in FIG. 4 to produce an MS array 140 as shown in FIG.3. The MS array is stabilized and packaged 320 according to thedisclosure hereof to produce a stability-packaged MS array 100. An MSsample 115 applied to the MS array 140 is tracked/indexed 310 byassigning a tracking identifier to the MS sample, and, in a samplerecord 240, associating the tracking identifier with a descriptor bywhich the MS sample can be distinguished from other MS samples on the MSarray. The sample record 240 is made available for use in associating360 offsite analysis results with tracking identifiers, and a datasystem 235 is optionally updated with the sample record 240. Thestability-packaged MS array 100 is dispatched 330 for transport 220, toan offsite analysis location 255 under the control of an analysisprovider 260, at which there is available an MS instrument 225 forperforming a desired MS analysis. The stability-packaged MS array isdepackaged 340 and an MS analysis 350 is performed on the MS array todetermine one or more MS characteristics of one or more of the MSsamples, whose tracking identifier(s) is/are determined by reference tothe descriptor contained in the sample record 240. The MScharacteristic(s) of a sample may be associated 360 with the trackingidentifier of the sample in a MS data record 230 and a data system 235may be updated with the data record 230. The MS characteristic(s) of theMS sample and associated tracking identifier are communicated 370 to acustomer 200 or other person entitled thereto. In some embodiments, thiscommunication may include providing a customer or other person entitledthereto with access to a data record including the MS characteristic(s),and/or a report 250 thereof, in response to a query 245 including thetracking identifier of a sample of interest.

In an exemplary embodiment of various of the systems and methodsdisclosed herein and illustrated in FIGS. 3 and 4, offsite MS analysisis performed for a customer 200, such as, for example, a pharmaceuticalcompany, that owns or controls a large sample library and is in need ofMS analysis which may be undesirable or impracticable to perform at thelibrary site 205. For example, the MS analysis may be of a naturerequiring instrumentation not available at the site where the library islocated, and/or requiring specialized expertise exceeding the customer'scapabilities. The customer may employ an analysis provider 260, whichmay be a person or entity having expertise in the type of MS analysisdesired, and having access to an MS instrument 225 of the type required.The analysis provider 260 optionally provides an arraying instrument 215at the site where the library is located, and performs the extracting ofMS samples from the sample library, the applying 300 of MS samples tothe MS array(s), the indexing/tracking 310 of the MS samples, thestability-packaging 320 of the MS array(s), and the dispatching 330 ofthe stability-packaged MS array 100 for transport 220 by a freightforwarder or other common carrier. Following the arrival of thestability-packaged MS array 100 at the analysis site 255, the analysisprovider 260 performs the depackaging 340 of the stability-packaged MSarray, the offsite MS analysis 350, and associating 360 the MS data withtracking identifiers. The analysis provider then provides the customerwith access to the MS analysis data, which is accessible by the trackingidentifiers previously associated with the MS samples.

In various embodiments offsite MS analysis may be performed by or for acustomer 200 or any other person or entity having need thereof. Variousaspects of the systems and processes disclosed herein may be carried outby a single person or entity, or by multiple persons or entities actingin cooperation and/or acting as independent providers or vendors. Insome embodiments, the offsite analysis 350 may be performed by, and/oran offsite analysis location 255 may be a facility owned or controlledby, an analysis provider 260, which may be a person or entity other thanthe customer 200 or other person or entity who owns or controls thesample library. In some embodiments, any or all of the systems andmethods disclosed herein, or any part thereof, may be performed by orunder the direction or control of a customer 200, an analysis provider260, one or more employees and/or third-party contractors under thedirection or control of either, or any other person or entity. In anembodiment, applying MS samples to an MS substrate 300, recordingtracking information 310, stability-packaging the MS array 320, anddispatching the stability-packaged MS array for transport 330 areperformed by or at the direction of a customer 200, and depackaging 340,MS analysis 350, associating results with tracking information 360, andcommunication of results 370 to the customer 200 or a designee of thecustomer are performed by or at the direction of an MS analysis provider260.

In some embodiments, a loading site 205 may be any location where sourcesamples are available from which MS samples may be extracted and appliedto MS substrates. Often samples will be obtained from a sample library.In an exemplary embodiment, library samples are stored in multi-wellplates or microtubes in a sample storage system providing temperatureand environmental control, and samples may be selected and extractedrobotically. The proximity to the sample storage system required forsamples to be practicably available for applying to an MS array maydepend upon a number of factors, such as, for example, the stability ofthe samples and analytes therein, the manner in which the samples arestored, availability of robotic or other facilities for handling andtransporting the containers, and security and access controlconsiderations. Thus in some embodiments, the loading site may be orinclude a room in which samples are stored in a sample storage system,or an adjacent or nearby room within range of practical sampleavailability. In some embodiments, samples may be obtained otherwisethan by selection from a stored library, such as, for example, directlyfrom a combinatorial chemistry process or from a chromatography or otherseparation process, or samples may be transported in well plates orother containers, in which case the loading site may be any location atwhich the samples are present and any required arraying instrument andany other resources required for applying MS samples to an MS substrateand stability-packaging the resulting MS array are available.

In some embodiments, an offsite analysis location 255 may be anylocation that is offsite from the loading site and where there isaccessible a MS instrument operable for performing the analysis ofinterest. In some embodiments, an offsite analysis location may be alocation at sufficient remove from the loading site so as to pose a riskof damage or alteration to the MS samples or substrate if the loaded MSsubstrate were transported to the location of the MS analysis instrumentwithout adequate stabilization and/or protection, taking into accountthe nature and composition of the samples and the conditions under whichthey are applied, transported, and/or analyzed. In some embodiments, theoffsite analysis location 255 may be in another building from theloading site 205. In some embodiments, the offsite analysis location isa facility owned or controlled by a person or entity, such as, forexample, an analysis provider, other than the owner of the samplelibrary.

Also disclosed herein are business systems, data processing systems, andmethods for offering and providing mass spectrometric analysis services.In embodiments, pre-loaded MS arrays may be received from or at thedirection of customers. These arrays may be loaded with MS samples by orat the direction of the customer at a suitable loading site, via anyloading modality such as, for example, acoustic deposition or any of theother loading modalities disclosed herein or otherwise regarded asacceptable by persons of skill in the art. In embodiments, the loadingsite is not located at the MS analysis provider's place of business andthe MS arrays are provided to the MS analysis provider with the samplesalready loaded on the array. The arrays may be stability-packaged asdisclosed herein. In embodiments, the arrays are delivered to theanalysis provider in analysis-ready condition, operable for performingMS analysis thereon without further sample preparation. The arrays maybe delivered to the MS analysis provider by the customer or by anysuitable delivery modality, such as by an independent carrier (e.g. acarrier not associated with the customer, or a carrier not associatedwith the MS analysis provider, or a common carrier). In embodiments, acustomer would also provide to the MS analysis provider a trackingrecord for each of the MS samples, associating a tracking identifierwith each MS sample, providing sufficient information whereby the MSanalysis provider can associate the tracking identifier with a specificphysical sample on the array, and providing any other information founduseful for an application of interest. For example, the tracking recordcould include the coordinates of a trackable locus at which the MSsample is deposited. In embodiments, the tracking record could beprovided to the analysis provider with the pre-loaded MS array, or couldbe communicated separately, such as by a network communication, or couldbe communicated in any other form or manner operable to provide to theanalysis provider sufficient information with which to identify andtrack the MS samples during analysis. In embodiments, the MS samples,the tracking identifiers, or both may be blinded. In embodiments, thecustomer does not provide to the MS analysis provider any informationfrom which the chemical structure of the MS samples can be determined,and/or from which other proprietary information that the customerdesires to avoid disclosing, such as, for example, the composition orprovenance of the analytes in the sample, can be determined. Inembodiments, the MS analysis provider performs MS analysis on an MSsample on the array, and associates data resulting therefrom with thetracking identifier of the MS sample in a data record. Typically thismay be done for 2 or more, or 10 or more, or 100 or more, or 1000 ormore MS samples present on the pre-loaded MS array, and the data recordsmay be incorporated in a data store such as a database. Access to thedata records and/or the data store may be provided to or at thedirection of a customer entitled thereto. The customer or otherauthorized recipient can then access the MS analysis data for each MSsample by the tracking identifier, and associate the analysis data withother data of the customer pertaining to the corresponding sample.

In an embodiment, it will be found useful in applying the MS samples tothe MS substrate to employ one or more application instruments 215, suchas, for example, an acoustic deposition instrument, and/or other devicesand laboratory instruments. Similarly, in obtaining MS samples from asample library or other source of samples, and/or in stability-packaginga MS array, various materials, devices and instruments may be used. Insome embodiments, any one or more of these materials, devices, andinstruments, including, for example, arraying instruments, MSsubstrates, mounting frames for MS substrates, and materials andapparatus for stability-packaging, may be owned and/or provided in wholeor part by a customer, or an analysis provider, or another person orentity. In an embodiment, the operations and methods for obtaining MSsamples from library samples or other sources, applying the samples toan MS substrate, stability packaging the MS array, dispatching the MSarray for transport, and related operations and processes, may beperformed by persons employed or contracted by or under the direction ofa customer, an analysis provider, or another person or entity.

FIG. 2 depicts schematically various general aspects of an embodiment ofa MS array 140, including a MS substrate 130, MS samples 115 applied toa sample accepting region 110 thereof, and an optional mounting frame135 in which the MS substrate may be mounted. Using the methods,apparatus, and compositions disclosed herein, MS arrays may be madehaving more than about 1,000, or more than about 2,000, or more thanabout 3,000 chemically distinct MS samples applied, representinganalytes of unknown compositions and potentially widely varyingproperties. Array features may be less than about 250 μm diameter, orless than about 100 μm diameter, and spaced less than about 800 μmapart, or less than about 400 μm apart.

A MS substrate may be of any composition, dimensions, and geometryoperable for application of a sample of interest thereto and MS analysisof the sample, and may include any of the MS substrates known to personsof skill in the art. In some embodiments, the MS samples are applied toa sample-accepting surface 110 of the MS substrate. In some embodimentsthe sample-accepting surface may be a substantially flat surface. Insome embodiments the sample-accepting surface may be porous, and/or maybe coated or doped. In some embodiments the MS substrate and/orsample-accepting surface may be composed in whole or part of one or moreof: a conductor (e.g. a metal); a semiconductor (e.g. doped silicon); aninsulator coated with a conductive/semiconductive material (e.g. coatedglass, plastic); a thin insulator on a conductive surface such as a 50um thick coverslip on stainless steel surface; or any other materialhaving or contributing to desired sample-application and/or MS analysisproperties.

In some embodiments as illustrated schematically in FIG. 2, a MSsubstrate 130 may be mounted in and/or affixed to a frame or mountingstructure 135. MS substrates may be fabricated or cut from the rawmaterial into numerous geometries. In some embodiments, the dimensionsand geometry of the MS substrate and/or the frame or mounting structureare compatible with an instrument for applying samples to the MSsubstrate, a MS analysis instrument, a laboratory robotics instrument,or other instrument with which compatibility is desired. In someembodiments, the dimensions and geometry of the MS substrate and/or theframe or mounting structure conform to or are compatible with a standardSociety for Biomolecular Screening (SBS) footprint, such as, forexample, 127.76 mm long by 85.48 mm wide(http://www.slas.org/education/standards/ANSI_SBS_1-2004.pdf), or astandard microscope slide dimension such as 75 mm long by 25 mm wide.Conforming to SBS geometries enables compatibility with a wide varietyof robotics, mass spectrometers, sample handlers, autosamplers, andother standard instrumentation. An advantage of mounting a MS substratein a mounting frame is that the dimensions of the MS substrate can bedetermined according to the number of samples and surface area requiredor other criteria of interest, while maintaining compatibility withother instruments via the frame or mounting structure, thereby allowingmore effective use of the substrate material.

In some embodiments, it will be found useful to employ as a MS substratenanoporous silicon coated with a liquid initiator that fills the pores(i.e. Nanostructure-initiator mass spectrometry, NIMS). In someembodiments an MS substrate may be prepared or treated according to anypreparation or treatment modality operable to produce desiredproperties, which may include any one or more of the MS substratepreparation or treatment modalities known to persons of skill in the artas suitable for the types of MS analysis techniques described in thisapplication. This may range from simple planar substrates including butnot limited to, silicon and other semiconductors, gold, steel, stainlesssteel, glass, polymeric materials especially conductive polymers,laminated materials etc. to more complex structured surfaces. A widerange of structured surfaces are used in mass spectrometry including butnot limited to, thin layer chromatographic surfaces, microfabricatedsilicon or other semiconductor surfaces, porous materials includingsilicon and other semiconductors and combinations of planar andstructured surfaces including those coated by other materials. Anexample of planar coated surfaces would be stainless steel coated withsinapinic acid or other MALDI matrix materials. By way of example of acoated structured surface, the MS substrate may be prepared generallyaccording to the protocol for producing NIMS surfaces as described inWoo H-K., Northen T. R., Yanes O., Siuzdak G. Nanostructure-InitiatorMass Spectrometry (NIMS): A protocol for preparing and applying NIMSsurfaces for high sensitivity mass analysis. Nature Protocols 3,1341-1349 (2008), which may be adapted to accommodate a desired chipgeometry and surface chemistry.

In some embodiments, coatings and/or sample additives may be employed,which may include any coatings or additives operable singly or incombination to improve spot placement, spot homogeneity, and/or spotreproducibility, and/or otherwise enhance the printed array quality; toimprove and/or facilitate ionization of sample; to improve any propertyof the mass spectrometry readout such as, for example, sensitivity,quantitation, or reproducibility; or to improve or modify any otherproperty or characteristic of interest. In some embodiments, forexample, a hydrophobic fluorous coating, as used in the production ofNIMS chips, may be employed to produce high contact angles (e.g. >90degrees) with aqueous sample droplets, which results in a MS sample spotthat covers a smaller (e.g. 250 microns or less or 100 microns or less)diameter. In some embodiments coatings and/or surface treatments areemployed to improve sample distribution homogeneity within a spot. Insome embodiments a degree of sample distribution homogeneity is obtainedsuch that independent samplings across a spot have a normalizedintensity coefficient of variation less than 50% for a known analyte inthe sample.

In various embodiments, MS samples 115 may be or include or be sampledor made from any one or more materials or substances desired to beanalyzed by mass spectrometry and capable of being applied to a MSsubstrate. In some embodiments an MS sample is, includes, or is derivedfrom an aliquot of a sample present in a combinatorial or other samplelibrary, such as, for example, a small organic molecule library,enzymatic reactions, drug libraries, libraries of biological materialsobtained from tissues, biofluids, or microorganisms, peptide libraries,protein libraries, polymer libraries, biopolymer libraries, librariesobtained from combinatorial chemistry approaches, and small moleculelibraries with one or more molecule conforming to Lipinski's Rule ofFive. These libraries may, for example, be composed of small molecules,natural and/or synthetic, such as used for the purposes of drugdevelopment, enzyme development or medical diagnostics. Libraries may bethe result of synthetic and/or combinatorial chemistry and/or beobtained from plant, microbial, or animals including human tissues andbiofluids. Libraries may also be composed in whole or part of activityassay mixtures that include proteins, co-factors, reaction substratesand/or other necessary components to assess activity, such as microsomesor cells.

Commonly, compound libraries used in, for example, drug development andresearch, may include a large number of individual samples, which maynumber in the thousands, tens of thousands, hundreds of thousands, ormore, with each sample separately stored. Often library samples aredissolved or suspended in a buffer or solvent, and disposed in wellplates or other containers known in the art, which are stored in acarefully controlled environment and handled and accessed robotically. Astored sample library may easily occupy a room-sized volume, part ofwhich comprises storage racks, associated environmental control systems,and robotic handling equipment that is not readily movable. Therefore,for a large library-derived sample set to be analyzed by MS techniquesnot available at the library location, it is of great practical utilityto substantially miniaturize the sample set, preferably in a way thatalso provides for maintaining stability and integrity of the samples.The methods, apparatus, and compositions disclosed herein may beemployed to prepare a miniaturized sample set wherein sample volumes arereduced from on the order of at least tens of microliters to on theorder of nanoliters, with samples representing the contents of largewell plates miniaturized onto readily transportable and stabilizablechips similar in size to standard microscope slides. The full massspectrometric informational content of a large sample set occupying onthe order of cubic meters of storage may be reduced to MS arraysoccupying less than 1/20 of the volume occupied by the source library,or less than 1/100 the volume, or less than 1/1000 the volume, or lessthan 1/10,000 the volume. In this way the packaging volume of a samplelibrary or subset thereof (that is, the volume required to be packagedif transport is desired, including the samples and their containers) maybe markedly reduced. In general, using the methods and apparatusdisclosed herein, the packaging volume required to package a samplelibrary or subset thereof for transport may be reduced from thatrequired for packaging multiple well plates or sample tubes to thevolume occupied by an MS array of dimensions as disclosed herein, for areduction in packaging volume of 20:1, or 100:1, or 1000:1, or 10,000:1,making practicable the transport of sample libraries would otherwise beso large and unwieldy for convenient packaging and shipping.

In some embodiments, MS arrays having very high total complexity areproduced, stability-packaged, transported, and/or offsite analyzed. Thecomplexity of an MS array may be expressed in terms of the number ofhyperspectral dimensions present, which may be computed as the massrange observed divided by the resolving power of the analysis, where themass range observed is the difference between the highest and lowestmasses observed, and the resolving power is the smallest differencebetween two masses that can be resolved as distinct peaks. Thehyperspectral complexity of an MS array may be expressed as the numberof hyperspectral dimensions for each spot or MS sample, summed over allthe MS samples present on the MS array. In general, as complexityincreases, the need increases for the precautions disclosed hereinagainst alteration and/or contamination of MS samples and associated MSsubstrate compositions, coatings, matrices, etc., while awaitinganalysis, since the information-degrading effect of any suchcontamination or alteration is likely to be relatively greater incomparison to the information present corresponding to thecharacteristics sought to be measured. The methods, apparatus,compositions, and articles of manufacture disclosed herein may be usedto produce, stability-package, transport, and/or perform and reportoffsite MS analysis on MS arrays having a total hyperspectral complexityof more than 10,000, or more than 100,000, or more than 500,000, or morethan 1,000,000.

In various embodiments MS samples applied to MS substrates may includeany one or more compositions and/or analytes capable of having an MScharacteristic measured or estimated by MS analysis according to any ofthe methods disclosed herein or known in the art. In some embodiments MSsamples may be, include, or be sampled or made from, for example, one ormore liquids, gases, solutions, emulsions, slurries, colloidal mixtures,gels, powders, suspensions of sample material or other suspendedentities, and/or biological fluids, such as, for example, blood, serum,cerebrospinal fluid, or urine. In some embodiments MS samples may besampled or derived from an organism, such as, for example, an animal, amicrobe, a plant, or a parasite; or from a tissue, tissue culture,bacterial culture, or other biological source, such as, for example, ahuman cell culture extract, a tissue extract, lysed cells, orhomogenized tissues. Analytes whose MS characteristics may be desired tobe analyzed by MS analysis, present in MS samples in variousembodiments, may include, for example, one or more analytes selectedfrom organic molecules, inorganic molecules, metals, ceramics, proteins,enzymes, biomarkers, transcription factors, membrane proteins,cytoskelatal proteins, peptides, polypeptides, nucleic acids, nucleicacid analogs, metallo-proteins, chemical catalysts, metallic groups,antibodies, cells, ions, ligands, substrates, receptors, biotin,hydrophobic moieties, alkyl chains, phenyl groups, co-factors, or anyother type or class of analyte at least one MS characteristic of whichis capable of being measured or estimated by MS analysis.

In some embodiments MS samples may optionally include or containsubstances having any chemical, physical, and/or biological propertiesdeemed useful, such as, for example, reagents, buffers, solvents,surfactants, wetting agents, lubricants, drugs, etc. In someembodiments, MS samples may include or have added thereto one or moreadditives such as organic alcohols (e.g. isopropanol, methanol) orpoly-alcohols (e.g. glycerol) or other organic solvents. Additives suchas, for example, the foregoing may be usefully employed to stabilize thesample droplets and improve dried array spot homogeneity and/orincreasing mass spectrometry detection quality/sensitivity and/orreproducibility. In some embodiments, such additives may include, forexample, a quantitation standard, a mass accuracy standard, a signal tonoise enhancer, a pH modifying additive, a solubility modifyingadditive, an ionization enhancing additive, a derivatization material, avolatility modifying additive, an adduction material, and/or a label,molecular bar code, or tag. In some embodiments, MS samples may bedissolved, suspended or mixed in, or otherwise associated with one ormore solvents, buffers, pure water, mixtures of water and organic, orother substances, such as DMSO. A solvent may be polar or non-polarand/or may be volatile, such as a solvent having a boiling point belowambient temperature and pressure, or may be non-volatile. In someexemplary embodiments it will be found advantageous to employ MS samplesdissolved, suspended, and/or mixed in DMSO, alcohol:water mixtures,and/or standard chemical buffers. In some embodiments an MS sample mayinclude and/or be derived or sampled from an extract, such as forexample an alcohol extract, a chloroform extract, an ether extract, aDMSO extract.

MS samples may be mixed with reagents to improve their desorption and/orionization and/or surface wetting characteristics. Common materials usedfor this purpose are photoreactive compounds including but not limitedto MALDI matrix materials, common examples include3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid),alpha-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid(DHB), picolinic acid. They may also include a wide range of acids orbases (e.g. trifluoroacetic acid, ammonium bicarbonate, etc.).Nanomaterials can also be used, such as, for example, goldnanoparticles, cobalt nanoparticles. Additives that can also be used toenhance surface wetting resulting in more even sample deposition includefor example colloidal particles, surfactants, and organic solvents.

A plurality of MS samples may be applied to an MS substrate to producean MS array. MS samples may be applied to an MS substrate by any methodor modality operable to produce on the MS substrate a deposition ofsample material capable of analysis therefrom by mass spectrometry andhaving volume, spot dimensions and spacing, and other characteristics assuitable for an application of interest. In some embodiments, MS samplesmay be applied to the MS substrate by application methods such as, forexample, contact printing, piezo printing, pipette printing, or ink jetprinting. In some embodiments it is preferable to apply MS samples tothe MS substrate using non-contact application methods such as acousticprinting, as described in further detail in the examples below, to avoidcontamination, and facilitate high-throughput processing and highdensity arraying. In some embodiments, MS samples applied to an MSsubstrate are immobilized and/or positionally constrained thereon in anymanner operable to produce an array of stability adequate for anapplication of interest. In some embodiments MS samples are immobilizedand/or positionally constrained by, for example, a chemical interaction,such as a covalent or ionic bond with the MS substrate or a coating orlinker; by an affinity interaction with the MS substrate or an affinityreagent; and/or by a physical interaction such as, for example,adsorption, trapping, or hydrophobic or van der Waals interactions.

In some embodiments, acoustic printing of samples onto the sampleaccepting surface will be found to produce superior results withhigh-throughput mass spectrometry readout of high-density sample arrays.In an embodiment as depicted schematically in FIG. 13, MS samples 115are applied to a MS substrate 130 by acoustic printing. The sampleaccepting surface 110 of the MS substrate is positioned facing the wellopenings of a film bottom well plate 400 or other container providing anacoustically transmissive path for acoustic energy. An acoustic horn 410emits an acoustic pulse into sample 405 present in the well orcontainer, causing a droplet of sample 425 to be ejected toward thesample accepting surface of the MS substrate. Actuators 415, 420 may beprovided to position the MS substrate and the well plate or othercontainer, respectively, relative to each other so as to deposit sampledroplets at predetermined positions on the MS substrate. In someembodiments the actuators are controlled by a computer and/or roboticstages are used so as to apply MS samples at predetermined positionsand/or in a predetermined pattern as specified in a computer file.Acoustic printing is a completely non-contact printing method thatutilizes an acoustic horn (transducer) 410 positioned below a reservoiror well (such as a microtiter plate 400) to emit a focused sound wavethat in turn ejects a droplet 425, typically in a volume/size range onthe order of tens of picoliters to tens of nanoliters. An advantage isthat the sample never contacts the printing apparatus, which eliminatesthe need for disposable print heads/tips or wash solvent. Thenon-contact nature of acoustic printing eliminates cross contaminationfrom the printing apparatus, which is important for high-sensitivitydetection such as mass spectrometry. An exemplary embodiment using ofacoustic printing for arraying of MS samples onto an MS array isdescribed in Example 7.

Sample pitch is determined by a range of factors including the MSsurface and MS sample physio-chemical properties, the volume of sampledeposited, and the sample deposition instrumentation, amongst otherfactors. For example, using the hydrophobic fluorous liquid coated NIMSMS samples for printing MS samples containing organic solvents willresult in lower contact angles and larger spot diameters than usingaqueous samples. In this case, the minimum pitch would be increased toprevent potential mixing of spots. Similarly, depositing larger samplevolumes will result in larger spot diameters and will result in largerminimum pitches to prevent spot mixing. In other cases the minimum pitchwill be limited by the capabilities of the deposition instrumentation.For example, a typical 8-span liquid transfer robot may have a minimumlateral resolution of about 500 μm and a minimum pipetting volume ofhundreds of nanoliters, limiting the pitch to about 500 The preferreddeposition method of acoustic transfer systems can print sub-nanolitervolumes with capabilities of sub-500 μm pitches. Printing of 1 nl orsmaller volumes of MS samples with contact angles of about 90 degrees orhigher using acoustic transfer system can achieve high density MS arraysthat most efficiently enable transport of large libraries. The resultinghigh density MS arrays typically require control environments to preventcross-contamination. Rehydration of the arrays during transport due tocondensation (e.g. cold conditions typical of air-shipping) combinedwith vibration can result in mixing of samples invalidating theexperimental results. Therefore, it will be found useful with the MSarrays to utilize the described shipping systems

In some embodiments and/or for some applications it may be found usefulin applying an MS sample to an MS substrate to apply a solid phaseanalyte or carrier, such as, for example, a frozen sample or fractionthereof, or a bead or nanoparticle. In some embodiments an MS sample isapplied in whole or part as a gas or aerosol, such as by a nebulizerprint head that nebulizes a liquid into a gas or aerosol that is thencaptured by the surface. Application of sample as a gas or aerosol mayoffer advantages including improved intra-spot homogeneity (sincenon-uniform drying effects are minimized), reduced inter-spot cross-talk(since the possibility of migration of liquid droplets is avoided), andpotentially improved signal-to-noise due to reduction of solventsuppression on the surface (volatile solvent evaporates faster thansample analytes, therefore nebulized solvent goes away into thesurrounding air while less volatile nebulized analytes are captured onthe surface as a sample spot).

In some embodiments and/or applications it will be found useful to applyan MS sample wherein a reaction or interaction of interest is occurringor has occurred. For example, in some embodiments an MS sample mayinclude any two or more interacting entities of interest such as, forexample, a protein interacting with a small molecule, a peptide, anoligosaccharide, a nucleic acid, another protein, or a cofactor; anenzyme interacting with a substrate; or a metabolite undergoing orproduced as a product of a reaction with another species. In someembodiments an MS sample includes two or more analyte entities that arecombined on the MS substrate, such as by sequential or simultaneousapplication of two or more compositions to the same MS substrate locus.In some embodiments a reaction or interaction occurring in an MS sampleis quenched, such as by applying a quenching substance and/or using anyof a number of methods common to the art, including, for example,temperature shifts, pH shifts, and/or addition of organic solvents tothe MS sample after a predetermined reaction or interaction time.

It being one of the objectives of the disclosure hereof to providemethods and apparatus whereby MS analysis can be performed on sensitiveand/or confidential samples at a location not under the control of theowner of the samples, while protecting the identity of the samples fromunauthorized disclosure or discovery, in some embodiments, MS samplesare blinded by applying them to the MS substrate in a manner and/or inquantities such as to prevent, impede, and/or make impracticable theanalysis of the MS samples by modalities or techniques other than theintended MS analysis. Thus an unauthorized person, even if in possessionof the MS array, would be able to obtain, at most, the informationobtainable by MS analysis, and would be unable to determine the exactchemical structure and/or identity of the analyte(s) present. In someembodiments, samples are applied at a center to center distance of lessthan about 1 mm, or less than about 500 microns. In some embodiments thevolume of an MS sample applied to a MS substrate is less than about 1μL, or less than about 500 nL, or less than about 100 nL, or less thanabout 10 nL, or less than about 1 nL or less than about 300 pL, or lessthan 100 pL, or less than about 10 pL. In some embodiments a MS sampleapplied to an MS substrate has an analyte concentration of less thanabout 10 mg/mL, or less than about 1 mg/mL, or less than about 100μg/mL, or less than about 10 μg/mL, or less than about 1 μg/mL, or lessthan about 100 ng/mL, or less than about 10 ng/mL, or less than about 1ng mL, or less than about 100 pg/mL, or less than about 10 pg/mL, orless than about 1 pg/mL, or less than about 100 fg/mL, or less thanabout 10 fg/mL, or less than about 1 fg/mL, depending in part upon theconcentration of an analyte of interest in a sample source. For example,an organic molecule in a combinatorial library sample may be present atrelatively high concentration, while a biomarker may be present in asample at very low concentration. A particularly challenging needaddressed by some embodiments as disclosed herein is the need formethods and apparatus operable to miniaturize, stabilize, and performoffsite analysis on samples wherein, depending on the sample sources andother considerations, the concentrations of an analyte of interest mayrange very widely. In some embodiments an analyte molecule to becharacterized by MS analysis present in a MS sample applied to a MSsubstrate has a mass less than about 300,000 Da, or less than about30,000 Da, or less than about 3,000 Da.

In some embodiments, by limiting the volume and/or analyte concentrationof the MS samples, the quantity of analyte material present in a samplemay be limited to a quantity as to which NMR analysis or other non-MSanalysis modalities are impracticable; by disposing the MS samples onthe MS substrate at high density and/or in close proximity, extractionof a sample for an unauthorized analysis is made more difficult orimpracticable. The practicability of reverse engineering analysis ofsamples depends upon the determination, effort, and resources applied,and a practicable blinding strategy should therefore aim for reasonabledeterrence commensurate with the likely risk. Currently NMR analysis ofsmall organic molecule samples of mass on the order of about 0.1 mg isgenerally capable of producing acceptable signal to noise ratios (for ¹Hacquisitions, which, coupled with MS analysis might reveal anundesirable level of information for reverse engineering), and,depending on the analyte and the instrumentation used, as little as 15μg may be sufficient. However, NMR analysis of a MS sample applied to aMS array would require removal of sample from the substrate, likelyresulting in some loss of sample. Thus in various embodiments, a usefullevel of deterrence can be achieved by limiting the total mass ofanalyte in each MS sample applied to a MS array to less than 0.1 mg, orless than 50 or less than 15 μg. Further deterrence can be achieved byarraying the MS samples in close proximity to each other. For spotscloser together than about 1 mm, or for even greater deterrence, lessthan about 0.5 mm, as disclosed herein, elution of a sample withoutcross-contamination from adjacent samples becomes more difficult andother means of removing the samples from the array would likely have tobe employed. Thus in various embodiments a useful improvement indeterrence against possible reverse engineering can be obtained byapplying MS samples at a separation of about 1 mm or less, and improvedfurther at a separation of about 0.5 mm or less, and still further at aseparation of about 0.25 mm or less. At extremely close featurespacings, the risk of cross-contamination between spots, particularly inthe presence of any moisture, humidity, or volatilization, increases,potentially raising a need for more careful environmental control duringhandling, packaging, and transport.

In some embodiments, a MS sample applied to an MS substrate is trackedand/or indexed to associate the MS sample with information by which itsidentity and/or provenance can be tracked by a person having authorityto do so. A MS sample may be tracked and/or indexed by any method ormodality and/or using any apparatus operable to maintain an unambiguousrecord or trace of the identity and/or provenance of the sample. In someembodiments, the identity of the sample is encoded and/or recorded via aunique tracking identifier associated with the sample. In someembodiments where it is desired to preserve confidentiality regardingthe identity of a MS sample, a tracking identifier should preferably bea non-informative identifier that does not expose to an unauthorizedperson information regarding the identity, nature, or provenance of theMS sample or from which these might be inferred. In some embodiments,for example, randomly assigned or encrypted identifiers may be employedas tracking identifiers. In some embodiments, a tracking identifier of aMS sample may include or be derived from a sample ID of a library samplefrom which all or part of the MS sample was obtained, or the MS surfaceID and x-y position on the array or other position descriptor, or may bean arbitrary identifier such as a random number or key assigned to theMS sample, in which case a record may be made associating the arbitraryidentifier with other information sufficient to allow a person entitledthereto to determine the identity and/or provenance of the sample, itscomposition, and/or another characteristic of interest. A trackingidentifier may be associated with another data item in any manneroperable to allow retrieval of the data item using the trackingidentifier, such as, for example, by creating or updating a record,which may be a computerized or electronic record, containing both thetracking identifier and the data item, or by employing the trackingidentifier as a key in a database, hash, or other data structurecontaining the data item as a value associated or capable of beingassociated with the key, or by incorporating, conflating, and/orencrypting the data item into the tracking identifier itself.

In some embodiments, it will be found useful in tracking and/or indexinga MS sample to apply the MS sample to a trackable position on the MSsubstrate, and/or to associate a descriptor of the position of the MSsample on the MS substrate with a tracking identifier of the MS sample.A trackable position on the MS substrate may be any position capable ofbeing individually identified and/or addressed, such as, for example, aposition whose coordinates relative to a known datum are known ormeasurable, or a position that can be determined from characteristics ofthe MS sample applied to the MS substrate or of the pattern of samplesor other MS samples therein. A descriptor of the position of the MSsample may include any descriptor providing information fordistinguishing an MS sample from other MS samples and/or addressing anoperation to a particular MS sample (such as, for example, making an MSmeasurement thereon, applying additional material thereto, or performinga quality control evaluation thereon). For example, in some embodiments,a descriptor of the position of an MS sample may include a distance,angle, or other metric of the position of the MS sample relative toanother known position, such as the position of a fiducial mark on theMS substrate or a frame in which it is mounted; or relative to one ormore other MS samples; or relative to a structural feature of the MSsubstrate or frame, such as, for example, an edge or corner. In anembodiment as illustrated in FIG. 5, a fiducial mark 150 is provided onthe mounting frame 135 in which an MS substrate 130 is mounted, and thefiducial mark serves as an origin of a coordinate system. A fiducialmark may be any fixed and detectable mark or feature useful forpositioning and/or aligning a MS substrate or mounting frame and/orfixing a position of any object, such as a MS sample, relative thereto.The respective distances 155 and 160 of a MS substrate position ofinterest 180 from orthogonal axes of the coordinate system uniquelyspecify the position of the MS substrate, and may be incorporated in aposition descriptor. In some embodiments where an acoustic depositioninstrument or other robotic arraying instrument or movable stage isemployed to position the arraying instrument relative to the MSsubstrate, the positions at which MS samples are applied may be obtainedfrom positional readouts from the instrument(s) and/or the instruments'associated software.

In some embodiments, a position descriptor includes the respectivedistances of the MS sample from two or more fiducial marks or from oneor more coordinate axis or other datum determined thereby, and/or thefiducial marks may be usefully employed to assist in alignment andregistration of the MS substrate or frame in a spotter, MS instrument,lab robot, or other instrument. Exemplary embodiments are described inthe Examples below. In some embodiments, a position descriptor mayinclude the position of an MS sample relative to other MS samples, and aparticular MS sample may be tracked or identified by, for example,oversampling, determining the pattern of spots on the MS substrate, andtracking or identifying the MS sample of interest by its position in thepattern. In some embodiments wherein a distinguishing MS characteristicof one or more MS samples is known, an MS sample may be tracked,indexed, and/or identified in whole or part by observing the presence orabsence of the distinguishing MS characteristic.

In an embodiment as depicted schematically in FIG. 6, wherein MS samplesare obtained from a sample library disposed in a well plate 165, MSsamples may be tracked and indexed by applying the MS samples in apattern having a known or determinable relationship with the positionsof the library samples in the well plate. Thus, for example, MS samples116, 117, 118, 119 may be arrayed on an MS substrate 130 in a patterncorresponding to the positions of the respective library samples 176,177, 178, 179 from which the MS samples are obtained. It will beapparent that an exact correspondence is not required, and that manyother mappings are possible, provided that a relationship exists wherebythe position of a MS sample on the MS array can be mapped back to itssource. Alternatively, a tracking identifier may include or be derivedfrom the positions of the library samples in the well plate, such as,for example, the row and column designators of samples in a standardwell plate having row and column markers 170 175, together with a plateidentifier.

In some embodiments, MS samples may be tracked or indexed based on anyproperty or characteristic operable for uniquely identifying a sample,such as, for example, by the presence of one or more tags or otherchemical or physical identifiers such as a molecular bar code or tagwhich may be detectable in a mass spectrum of the sample. In someembodiments, for example, MS/MS fragmentation may produce afragmentation pattern from a spiked internal standard or analyte givinghighly specific identification of a sample spot. MS samples may also beindexed or tracked based on an orthogonal analysis system, for exampleabsorbance or fluorescence or phosphorescence or radiotracers. Forexample, inclusion of a fluorescent dansyl dye in known MS arraypositions would enable a rapid fluorescent image to map out the arrayfor subsequent MS analysis.

In an embodiment, as illustrated schematically by way of non-limitingexample in FIG. 1, there is provided a stability-packaged MS arrayincluding an MS substrate 130, having a plurality of MS samples 115applied thereon, an enclosure 100 surrounding the MS samples, and abarrier 105 isolating the MS samples from physical contact. In someembodiments a stability-packaged MS array further includes anenvironmental control system 125 disposed and configured to conditionthe environment within the enclosure, or at least the environmentimmediately proximate to the MS samples.

In an embodiment as illustrated in FIG. 1, a stability-packaged MS arrayincludes an enclosure 102 surrounding the MS samples. The enclosure maybe of any material, composition, dimensions, geometry, and/orconfiguration operable to establish a region surrounding the MS sampleswherein the environment to which the MS samples are exposed may becontrolled. Care should preferably be taken that any portions of theenclosure, and any other components of the packaging exposed to the MSsamples or the environment in which they are enclosed, are composed ofmaterials that do not emit vapors, gasses, particulates, or othercontaminants and are maintained free of any such materials orcontaminants and/or cleaned to remove them. In some embodiments asillustrated in FIG. 1, the enclosure 102 may be a sealed enclosure, suchas a Mylar bag sealed with a heat seal 132, enclosing the entire MSsubstrate, the MS samples, and optionally other components. In someembodiments the enclosure may be sealed over or against the MS substrate130 and/or frame or mounting structure 135. In some embodiments theenclosure may be wholly or partially impermeable and/or impervious togasses and/or vapors. Indicators may be included on or in the package toindicate mistreatment during shipment, which may in various embodimentsinclude any device, material, or component operable to disclose theoccurrence of a condition of interest, such as, for example MonitorMarksavailable from 3M which provide a visual history of time/temperatureexposure [solutions.3m.com/wps/portal/3M/en_US/Microbiology/FoodSafety/product-information/product-catalog/?PC_7_RJH9U523003DC02357P92O3O87000000_nid=NFNLL5PG88beX2JZNTZSLTgl].

In some embodiments the enclosure may be fully or partially evacuated.In some embodiments the enclosure may be charged with a non-interactivegas or fluid, which may preferably be of a composition that does notreact chemically with or otherwise contaminate or alter an MS sample orthe MS substrate in a manner that would introduce an artifact orinaccuracy in a MS analysis performed on the MS sample. In someembodiments a non-interactive gas or fluid may be or include an inertgas, such as, for example, argon, or a gas that is not chemicallyreactive with an MS sample, such as, for example, ultra-high purity(UHP) nitrogen. It will be apparent to persons of skill in the art thatthe reactivity of a particular gas or fluid with an MS sample may dependupon the composition of the MS sample, and selection of an appropriatenon-interactive gas or fluid should be guided by the known chemical andphysical properties of the gas or fluid and the MS sample. In general,and in many embodiments, it will be found useful to avoid or reduceexposure of the MS samples and/or MS substrate surface to moisture,humidity, particulates, solvents and their vapors, and contaminants,since these may mix, react chemically, or induce a state change in theMS samples or MS substrate, and/or may cause MS samples to spread,interact and/or cross-contaminate with other nearby MS samples, adsorbcontaminants from the surrounding air or gas, and/or change spotmorphology. Contamination can occur from the contact of the MS surfacewith gas, liquid, or solid materials that affect or interact with ordeposit materials onto the mass spectrometry surface and/or MS samples.An example of gas phase contamination is the adsorption of smallmolecules from the air by the nanostructure-initiator surface reducingthe performance of the assay. Gas and liquid phase contamination may,for example, occur in situations where hygroscopic materials printedonto the surface absorb water vapor or where water condensation formsdirectly onto the surface; in both cases the water can mix spots,degrade analyte, and be otherwise detrimental to assays. Exposure tooxygen, high temperatures, and light can similarly degrade samples andshould preferably be avoided. The integrity of the mass spectrometrysurface and printed samples may be preserved by utilizing the packagingmethods and modalities disclosed herein to minimize these types ofenvironmental exposures.

In an embodiment as illustrated in FIG. 1, a stability-packaged MS arrayincludes a barrier 105 isolating the MS samples from physical contact.The barrier may be of any composition, dimensions, or geometry anddisposed in any manner or position operable to protect the MS samplesfrom physical contact, such as, for example, physical contact with thebarrier itself, physical contact with the enclosure, and/or physicalcontact with any other object that might disturb or contaminate the MSsample or MS substrate. In some embodiments a barrier is disposed andconfigured to cover the MS samples and establish a space or gap 107between the MS samples and the barrier. It will be apparent to personsof skill in the art that many arrangements and configurations arepossible interposing one or more barriers covering, surrounding, and/ordisposed adjacent to one or more MS samples. In some embodiments, thebarrier may be integral with the enclosure. In some embodiments asillustrated schematically in FIG. 1, the barrier may comprise or be partof a container within which the MS array is disposed and the containermay be disposed within the enclosure 102. In some embodiments thebarrier may be rigid or semi-rigid, and/or held in position and/ordisplaced from the MS samples by a rigid or semi-rigid support. In someembodiments as illustrated in FIG. 1, the MS substrate with MS samplesarrayed thereon may be disposed within a container wherein the MSsubstrate and or frame or mounting structure on which the MS substrateis mounted are constrained in position by one or more positioningmembers 120, or in any other manner operable to position the MS samplesrelative to the barrier, thereby establishing a gap or space between theMS samples and/or MS substrate surface and a barrier 105 disposedadjacent to and/or covering the MS samples and/or MS substrate. In someembodiments, an MS substrate with MS samples arrayed thereon may bedisposed in a container such as, for example, a standard semiconductorwafer carrier or equivalent, which may optionally be modified asappropriate to accommodate the dimensions and geometry of the MSsubstrate and/or frame or mounting structure in which the MS substrateis mounted, and to ensure the maintenance of a gap or space adjacent tothe MS samples. In some embodiments, the semiconductor wafer carrier orequivalent may be disposed within the enclosure 102.

In some embodiments, a light shield is provided to protect the MSsamples and/or MS substrate surface from exposure to light. A lightshield may be of any composition, dimensions, and geometry and disposedin any position or manner operable to block or reduce the exposure ofthe MS samples and/or MS substrate surface to all or part of thespectrum of visible, infrared, and/or ultraviolet light. Whether a lightshield should be included in an embodiment, and if so the optical andother properties of the light shield may be determined based upon the MSsamples and substrate composition to be protected, and their sensitivityto deterioration and/or alteration by the various wavelengths of lightto which the stability-packaged MS array may be exposed. However,keeping in mind that an object of the disclosure hereof is to facilitatearraying and MS analysis of large numbers of distinct samples havingundetermined optical properties that may vary widely over the samplesbeing arrayed, it will usually be found preferable to shield the MSarrays completely from light. In some embodiments, the enclosure, thebarrier against physical contact, or another component of the package MSarray may be composed of or include an opaque or non-light transmittingmaterial or coating or otherwise integrate a light shield.

In some embodiments, an environmental control system 125 is provided. Anenvironmental control system may be or include any substance, material,device, or component operable for conditioning and/or modifying theatmosphere and/or environment within the enclosure 102, and inparticular the atmosphere and/or environment immediately proximate tothe MS samples and/or MS substrate surface, in a desired manner. In someembodiments, an environmental control system may include a dehumidifyingcomponent such as, for example, a desiccant. Avoidance of moistureduring transport and storage is particularly important as rapid changesin temperature/humidity (i.e. during airline cargo transport, or onremoval from cold-storage) can cause rehydration of the array spots dueto absorption of moisture from the air by the array spots or in caseswhere the sample is shipped at reduced temperatures for enhancedstability for example on dry or wet ice. Rehydration can increase therate of sample degradation, cause neighboring array spots tocombine/cross-talk, or generate sample spot inhomogeneity duringrepeated spot rehydration/drying that increases mass spectrometryreadout variability. In some embodiments an environmental control systemmay include a temperature controller disposed and configured to maintainthe temperature to which MS samples are exposed within predeterminedlimits. For example, the MS array and enclosure and protective barriermay be further enclosed in a temperature controlled box or package. Insome embodiments an environmental control system may include one or morefilters or other purifiers, which may be active or passive, operable topurify and/or remove or sequester contaminants such as, for example,particulates, moisture, and/or volatilized solvents or other undesiredsubstances.

Care should preferably be taken to avoid contamination of the MS samplesand/or MS substrate surface by particulates or other contaminants thatmay be emitted by any component of the apparatus, such as, for example,by an environmental control system or a component thereof such as, forexample, a desiccant. In some embodiments, an environment control systemincludes a desiccant composed of a contaminant suppressant compositionand/or packaged in a contaminant suppressant package. In someembodiments, a filter is provided and disposed to protect the MS samplesand/or MS substrate surface from particulates, while allowing passage ofgases and vapors. In some embodiments, the MS substrate and MS samplesare enclosed in a partially sealed or selectively permeable containerand a desiccant or other potentially contaminant-emitting component isdisposed outside the container, thereby allowing diffusion of gases orvapors from the container but inhibiting the migration of particulatesor contaminants into the container by substantially restricting theavailable paths and eliminating straight line paths by whichparticulates might enter the container. In some embodiments, wherein thecontainer is a semiconductor wafer carrier or equivalent, and theenvironmental control system is or includes a packaged desiccant, thecontainer may be partially closed and the packaged desiccant may betaped or otherwise disposed against a surface of the container.

In some embodiments, a stability-packaged MS array is de-packaged byremoving the MS substrate with MS samples applied thereto from theenclosure, protective barrier, environment conditioning system, and/orother packaging, and MS analysis is performed. In various embodiments,MS analysis and/or MS measurements may be performed by any MS techniqueoperable to provide a measure of one or more MS characteristics ofinterest. In some embodiments, the results of an MS measurement and/orMS analysis may include a mass spectrum and/or any part thereof orcharacteristic or quantity derivable therefrom, such as, for example, aplot or vector of peak intensities at one or more m/z values ofinterest, or a table of ions and their corresponding intensities. Invarious embodiments, a MS characteristic measured and/or reported mayinclude any characteristic or property of an MS sample capable of beingmeasured or estimated by one or more MS techniques, such as, forexample, a peak height or intensity at a specified m/z; the presence,absence, and/or abundance of a specified analyte or moiety in a MSsample; or the degree to which a sample matches a pattern or standard.

The methods, apparatus, compositions, and articles of manufacturedisclosed herein will be found useful in connection with offsite MSanalysis by any MS analysis method or modality and using any MSinstrument(s) operable for determining an MS characteristic of an MSsample applied to an MS array, such as for example, time-of-flight,magnetic sector, quadrupole, ion trap, ion cyclotron resonance,orbitrap, ion mobility spectroscopy, electrostatic sector analyzers, orhybrids of any of these mass analyzers.

In some embodiments, spatially defined desorption and/or ionizationapproaches are used to generate ions from the MS array such that thecomposition of array element(s) can be linked to the position of thearray element(s). Commonly this may be accomplished by scanning and/ortargeting a focused beam over the surface to desorb molecules and/orions from a defined region of the surface. However, while not common inthe art, it is also possible to desorb molecules/ions from larger (x-y)regions and maintain the relative x-y positions and then detect the ionsusing a spatially defined detector. Here the mass may be determinedusing a mass analyzer, for example time-of-flight, and the position maybe determined based on the detector element. There are a wide range oftechnical approaches for generating ions in a spatially defined manner.Many approaches simultaneously desorb and ionize the molecules.Approaches using focused beams for spatially defined mass spectrometryinclude: secondary ion mass spectrometry (SIMS), which utilizes an ionbeam to desorb and ionize molecules; MALDI, which utilizes a laser beam;desorption electrospray ionization (DESI), which utilizes a focusedelectrospray beam; and nanoDESI, which uses a small pool of solvent todetermine the locus from which sample is ionized. In some embodimentsmethods may be used in which the desorption and ionization processes aredecoupled, such as, for example, laser ablation electrospray ionization(LAESI) where an IR laser is used for desorption followed byelectrospray ionization, and liquid extraction surface analysis (LESA),where the samples are extracted from a surface and then analyzed fromthe liquid phase typically using electrospray ionization. In someembodiments, methods may be used wherein ions can be generated on thesurface first (e.g. by adding acidic buffer) and then desorbed using aspatially defined desorption technique.

In some embodiments, MS analysis is performed by laserdesorption/ionization (LDI) MS, wherein laser energy from, for example,an ultraviolet laser or an infrared laser, is applied to desorb analytesfrom a surface and volatilize and ionize them making them available tothe ion optics, mass analysis, and/or detection components of a massspectrometer. In some embodiments MS analysis is performed by matrixassisted laser desorption/ionization (MALDI), wherein a matrix isemployed to absorb energy from the laser and facilitatedesorption/ionization. In various embodiments a matrix may be or includeany material or composition operable to absorb laser energy and/orcontribute to ionization and/or desorption of analyte, such as, forexample, nanoparticles or colloids (e.g. gold nanoparticles), and/ororganic matrix molecules e.g. alpha-cyano-4-hydroxycinnamic acid,dihydroxy benzoic acid (DHB), and/or sinapinic acid, which may beapplied to, for example, indium tin oxide glass slides or steel/goldtargets. In some embodiments MS analysis is performed by surfaceenhanced laser desorption/ionization (SELDI), wherein analyte moleculesare captured or bound, by for example affinity capture or covalentbinding, onto a MS substrate surface. In various embodiments of LDI,matrix may be applied in any manner operable to produce a desiredmatrix-analyte composition on an MS substrate, such as, for example, byapplying matrix to an MS substrate surface prior to application of MSsample; applying a mixture of matrix and sample; applying matrix andsample simultaneously or sequentially in any order and allowing them tomix on the substrate, such as by sequential acoustic deposition ofmatrix and sample onto the same substrate locus; or applying matrixafter application of the MS sample to the substrate and allowing thematrix and sample to co-crystallize onto the substrate. In someembodiments, any other type of LDI MS operable for a desired analysismay be employed to perform MS analysis on an MS array, such as, forexample, surface enhanced neat desorption (SEND), or surface enhancedphotolabile attachment and release (SEPAR). The presence of matrixpotentially poses additional stability concerns and underscores theusefulness of stability-packaging according to the methods and apparatusdisclosed herein where offsite analysis is contemplated.

In some embodiments, particularly where signals of interest lie in arange below about 700 Da m/z where background signals from matrixentities may be problematic, matrix-free LDI techniques may be employed;these may include any matrix-free technique operable for MS analysis ofa MS sample or analyte of interest, such as, for example, use ofsubstrates including light-absorptive materials such as nanoporoussilicon, silicon nanowires, gold nanoparticles, or otherenergy-absorbing materials; nanoparticle-assisted LDI MS (nano-PALDIMS); sol-gel assisted LDI (SGALDI); and matrix free material-enhancedLDI (MELDI). In some embodiments, light can be used to desorb the sampleas in, for example, laser ablation electrospray ionization (LAESI), orthe sample array may be scanned using direct analysis in real time(DART) techniques or DESI. In some embodiments combinations of thedescribed approaches may be employed, such as, for example, NIMSfollowed by photoionization to improve ion yields. Many variations,embodiments, and combinations of techniques for MS generally and LDI MSin particular exist, and are operable for use with MS substrates and MSarrays of many types and compositions, and the methods, apparatus,compositions, and articles of manufacture disclosed herein may usefullybe employed to apply MS samples to such substrates and/orstability-package MS arrays for offsite analysis by any of such methods.

The methods disclosed herein will be found particularly useful inembodiments wherein factors or conditions are present suggesting a needfor precautions to ensure the integrity of the MS samples and MS arrayduring handling and transport, such as, for example, where MS samples,MS substrates, coatings, or matrix include compositions that areparticularly susceptible to absorption of moisture, volatilization, orcontamination; where MS samples are applied at a spot spacing or on asurface posing greater risk of cross-contamination between samples dueto inadvertent solvation or other factors; or where the association ofthe MS samples with the MS substrate is relatively weak. Thus, forexample, the methods disclosed herein will be found particularly usefulin connection with non-capture application techniques, wherein MSsamples are not captured on the MS substrate, such as by covalentlybinding to the MS substrate, capture via strong affinity tags, and/orother modalities that substantially immobilize or stabilize MS sampleson the MS substrate.

In some embodiments one or more quality control tests or measurementsare performed. A quality control test or measurement may comprise anytest, measurement, or observation for verifying that an operation orstep as disclosed herein succeeded within specified bounds and/orproduced a result conforming to a standard or tolerance of interest.Quality control checks can be usefully employed with respect to anyoperation and at any stage of the methods disclosed herein. Inparticular, it may be found useful, for example, to verify during orafter the application stage that MS samples are applied in volumes, atloci, and/or having morphology as intended; to verify that an MS arrayhas been stability packaged correctly and that all components of thestability package are present, correctly disposed, and functional; thata MS array remains stable and uncontaminated following packaging andtransport; and/or that measured MS characteristics conform to expectedranges or standards. In some embodiments, quality control includesinspecting an MS array after application of MS samples thereto, whichmay include observing or measuring any quality control property ofinterest, such as, for example, the area occupied by an MS sample, themorphology of an applied MS sample, the light reflective or absorptiveproperties of an applied MS sample, the fluorescence properties of anapplied MS sample, the spot-to-spot variability of any property, or theuniformity of any property over the MS array, from spot to spot, orwithin a spot. In some embodiments quality control testing may includevisual inspection and/or spectroscopic analysis of the MS array. In someembodiments it will be found useful to include on an MS array one ormore MS samples comprising quality control standards, upon which MSmeasurements may be made so as to verify the continued stability andcontaminant-free state of the MS array at the time of MS analysis. Suchstandards may comprise any composition operable for MS analysis thereonand having an MS characteristic capable of measurement; in someembodiments it will be found useful to employ standards havingcomposition similar to MS samples and/or potentially susceptible todetectable alteration in the presence of particular possible confoundingfactors. Thus, for example, in an embodiment where cross-contaminationbetween MS samples due to moisture absorbance is a concern, a pair ofclosely adjacent MS samples could be employed comprising compositionshaving distinguishable MS characteristics and known to be susceptible tomoisture absorbance and instability therefrom, so that intermixing couldbe detected on MS analysis. One or more quality control standards may beemployed to verify the integrity of the stability packaging, such as,for example an indicator that produces a detectable signal in thepresence of moisture or a contaminant. Standards and/or quality controlchecks may also be usefully employed as appropriate to the MS analysismethod used, as will be familiar to persons of skill in the art.

In some embodiments, methods and/or apparatus are provided forrecording, maintaining, transmitting, and/or providing access totracking information and MS analysis results. Useful aspects may includeone or more of: assignment of tracking identifiers to MS samples;recording of descriptors of positions at which MS samples are present onan MS array; recording the tracking identifiers of MS samplescorresponding to position descriptors; communicating tracking and/orposition descriptor information to a MS analysis provider; employingposition descriptor information in performing MS analysis and targetingparticular samples; associating MS results with particular samplesand/or their position descriptor information and/or their trackingnumbers; and/or communicating MS results with associated trackinginformation to a customer or person entitled thereto, in bulk, inresponse to a query including the tracking information, or otherwise.Information may be recorded, processed, updated, queried, andcommunicated in any manner and using any modalities operable forinformation processing, Information may be recorded on paper, onportable electronic media such as a magnetic disk, optical disk, memorycard, USB memory device, or in the memory of a computer, or in any othermodality operable to store data. Information may usefully be organizedin records and/or in a data structure that facilitates efficientsearching, updating, querying, or other operations of interest, such as,for example, a relational database, an object-oriented database, an XMLor other structured record, or a table-based database or data structure.

A data management system can be used to link index locations of thesamples on the MS substrate to resulting spectral data. This can beachieved by generating a file containing the positions of the samplesand their relative position and mapping this information to the spectradata based on the relative position. In an embodiment the index file isgenerated for the sample deposition and is transferred to a computersystem and upon analysis of the printed samples at a remote location,the resulting spectral data files are then correlated with the originalindex system such that individuals can use the spectra data to obtaincompositional information on one or more indexed sample.

In an embodiment, information including tracking information such astracking identifiers and MS analysis results pertaining to thecorresponding MS samples may be communicated to a customer or otherperson entitled thereto by any method and/or modality operable totransmit the desired information, including, for example, providing awritten report; providing data on non-transitory computer readablemedia; and/or providing access to a data management system operable torespond to a query including tracking information by outputting the MSanalysis results pertaining to the corresponding MS samples. In anembodiment a customer may record tracking identifiers corresponding toeach MS sample in its own data system at the time of providing thesamples for applying to MS arrays, and MS analysis results are providedon non-transitory computer readable media in a structure and formatcompatible with a customer's data system, or in a standard structure andformat such as XML, so that the customer may readily incorporate the MSanalysis results in its own data system.

In an exemplary embodiment of a data management system as illustrated inFIG. 14, a customer 200 selects library samples desired to be analyzedand provides a record 375 including, for each sample, a trackingidentifier and a sample locator, which may be any information by whichthe specified sample may be located and obtained for purposes ofextracting an MS sample therefrom, such as, for example, an identifierfor accessing a specific sample in a sample management system, or a wellplate number and row/column. In an embodiment, the sample locator may beemployed as a tracking identifier. The customer maintains recordsassociating each tracking identifier with the corresponding librarysample. MS samples are extracted from the library samples and applied300 to an MS substrate. A record 380 is created including, for each MSsample, the corresponding tracking identifier and a position descriptorindicating the position of the MS sample on the MS substrate. Theinformation of the record 380 is used to control a MS instrument toselect a specified MS sample for analysis. In an embodiment, the record380 may be included in a non-transitory machine readable file that canbe employed directly or used to generate another file compatible withthe MS instrument. In an embodiment, the record 375 and/or the record380 may be stored in a data management system 235 where they may beaccessed by the customer, an MS analysis provider, or another personentitled thereto. From performing offsite MS analysis 350, MS analysisresults for an MS sample are associated with the tracking identifiercorresponding to the MS sample in a record 385, which is added to a datamanagement system 235. The customer may then submit a query 390including the tracking identifier corresponding to a sample of interestto the data management system and retrieve therefrom a record 395including MS analysis results for the MS sample to which the queriedtracking identifier corresponds. In an embodiment, a data managementsystem may be implemented in any manner and using any apparatus operableto carry out the desired operations, such as, for example, by a computerrunning database software to which records are added and from whichdesired information may be queried. In an embodiment, user interfacesmay be provided for entering records and/or queries and/or fordisplaying information. In an embodiment, access is provided whereby acustomer, MS analysis provider, or other authorized person may accessthe data management system remotely over a network, such as, forexample, a local area network, or the internet.

MS samples and MS substrates may be prepared, handled, manipulated,combined, transformed, analyzed, and/or processed according to any ofthe many methods, techniques, best practices, guidelines, and heuristicsand using any of the devices, components, apparatus, or structuresdisclosed herein or known to persons of skill in the art. The datamanagement methods disclosed herein may be implemented using anyoperable methods and devices, in software or hardware, using singleprocessor systems and/or multiprocessor and/or distributed systems,allocating tasks among components, software or hardware modules, andprocess steps in any manner, and performing tasks in any order, operableto carry out the disclosed tasks.

Also disclosed herein is a method of stability-packaging a MS substratehaving a plurality of MS samples applied thereto, the method includingenclosing the MS samples in an enclosure, which may be a sealedenclosure; establishing a contaminant-protective environment proximateto and/or surrounding the MS samples; and protecting the MS samples fromphysical contact. In some embodiments establishing acontaminant-protective environment may include any measures and/oremployment of any materials and/or components operable to block, filter,remove, or otherwise restrict or prevent the presence of one or morecontaminating substances in the environment proximate to the MS samples.Contaminating substances may include any substances capable of alteringor affecting an MS sample in a manner posing a risk of introducinginaccuracies into a MS measurement. The particular contaminatingsubstances to be protected against, and the degree of protectionrequired, may depend upon factors such as the composition of the MSsamples, the MS substrate and any coatings or matrix present, thetemperature, pressure, humidity, vibration, and other conditions towhich the stability-packaged MS samples may be subjected, and any otherfactors affecting the susceptibility of particular MS samples toalteration or contamination. In some embodiments, contaminatingsubstances of potential concern include moisture and/or particulates. Insome embodiments establishing a contaminant-protective environmentincludes establishing a substantially dehumidified, particulate-freeenvironment.

The MS samples may be enclosed in an enclosure as disclosed herein or inany other manner operable to establish or define a region proximate toand/or surrounding the MS samples wherein a substantially dehumidified,particulate-free environment may be established. In an embodiment, theMS samples may be enclosed in a Mylar bag or sleeve and the opening(s)in the bag or sleeve may be closed by heat sealing, sonic welding, useof an adhesive, clamping, or any other method for operable forestablishing and maintaining a seal. In some embodiments a sealedenclosure may be provided and sealed according to any method and/orusing any materials and/or components operable to provide a sealedcompartment proximate to and/or surrounding the MS samples.

Establishing a contaminant-protective environment proximate to and/orsurrounding the MS samples may include fully or partially evacuating anenclosure within which the MS samples are enclosed; charging anenclosure within which the MS samples are enclosed with a substantiallymoisture-free, particulate-free gas or fluid, which may be an inert ornon-interactive gas or fluid as disclosed herein; and/or dehumidifyingand/or filtering or removing particulates from a gas or fluid proximateto the MS samples. In some embodiments, a method of packaging a MSsubstrate having a plurality of MS samples applied thereto may includedisposing a desiccant or other conditioner in diffusive communicationwith a gas or fluid proximate to or surrounding the MS samples.

Protecting the MS samples from physical contact may include disposing aphysical barrier covering and/or proximate to the MS samples asdisclosed herein, or may be accomplished in any other manner operable toprotect the MS samples against contact with a foreign object.

Also disclosed herein is a method of packaging, according to thedisclosure hereof, an MS substrate having a plurality of MS samplesapplied thereto, and dispatching the stability-packaged MS array foroff-site transport. In an embodiment, dispatching a stability-packagedMS array for off-site transport may include directing, arranging for,contracting for, giving instructions for, or otherwise causing transportof the stability-packaged MS array to a second location removed from alocation at which the MS array was packaged. In an embodiment,dispatching a stability-packaged MS array for off-site transport mayinclude, for example, depositing the stability-packaged MS array in themail; sending a stability-packaged MS array by messenger, or by afreight forwarder, package express service, or other common carrier; orhand carrying a stability-packaged MS array.

Also disclosed herein is a method including packaging, according to thedisclosure hereof, an MS substrate having a plurality of MS samplesapplied thereto, and receiving information comprising a MScharacteristic, determined by MS analysis performed at an off-sitelocation on at least one of the MS samples.

Also disclosed herein is a method of doing business including receivinga stability-packaged MS array from a customer, performing MS analysis onan MS sample present on the MS array, and communicating to the customerthe results of the analysis. In an embodiment, the method includesperforming MS analysis on an MS sample to which analysis is targetedusing a position descriptor associated with a tracking identifier, andcommunicating the results of the analysis to the customer in associationwith the tracking identifier, and information regarding the identity,composition, and provenance of the analyte(s) present in the MS sampleis not provided by the customer. In an embodiment, the method may alsoinclude any or all of applying the MS sample to the MS substrate,stability-packaging the MS array, dispatching the stability-packaged MSarray for offsite transport, and/or transporting the stability-packagedMS array.

Also disclosed herein are substrates, methods, and kits having a rangeof applications, and based on the ability to detect, quantify, orisolate analyte using desorption ionization mass spectrometry.

Provided herein in one embodiment is a substrate comprising a poroussemiconductor treated with a fluorous initiator and a photoactivecompound, which treatment enhances the ionization and desorption ofsamples deposited on its surface thereby enhancing the detection of thesample by desorption ionization MS analysis. The porous semiconductorprovided herein absorbs electromagnetic radiation which is used toionize the analyte that rests upon or is adsorbed on the substrate. Inone embodiment, provided herein is a substrate comprising a poroussemiconductor, a fluorous initiator adsorbed to the semiconductor and aphotoactive compound containing a fluorous group adsorbed to thesemiconductor. In another embodiment, provided herein is a substratecomprising a porous semiconductor, a fluorous initiator, and aphotoactive compound containing a fluorous group. The fluorous initiatoris a molecule that adsorbs onto or coats the substrate and in certaincases, adsorbs onto and/or coats the recesses of the porous substrate,and is believed to thereby trap or sequester the analyte that rests uponor is adsorbed on the substrate. The fluorous initiator vaporizes uponirradiation of the substrate (for example with a laser or ion beam) andis believed to facilitate the desorption of the analyte.

In certain embodiments, the semiconductor is selected from silicon,doped silicon, aluminum, polymeric resins, silicon dioxide, dopedsilicon dioxide, silicon resins, gallium, gallium arsenide, siliconnitride, tantalum, copper, poly silicon, ceramics, and aluminum/coppermixtures. In yet another embodiment, the semiconductor is selected fromsilicon, doped silicon, silicon dioxide and doped silicon dioxide. Inyet another embodiment, the semiconductor is silicon or doped silicon.In one embodiment, the semiconductor is a boron-doped p-type silicon. Inanother embodiment, the semiconductor is phosphorous doped n-typesilicon. In yet another embodiment, the semiconductor is arsenic-dopedsilicon.

As used herein in relation to porous semiconductor substrates, the term“porous” means having “pores”, or having recesses or void spaces. Incertain embodiments, the recesses are channels, wells or pits. Therecesses may have a random or ordered orientation or pattern. In certainembodiments, the recesses or void spaces have a degree of irregularity.The semiconductor may be rendered porous by any chemical and physicalmethods known to those of ordinary skill in the art, including etching,drilling and scratching. The semiconductor may be rendered porous byother methods including sintering, lithography, sputtering, sol-gelpreparation and other methods known to those of ordinary skill in theart. In certain embodiments, the porous semiconductor substrate haspores having a degree of irregularity mostly having a diameter of about5 nm to about 20 nm. In certain embodiments, the porous semiconductorsubstrate has pores having a degree of irregularity mostly having adiameter of about 10 nm.

In certain embodiments, the porous semiconductor substrate comprises afluorous initiator. In one embodiment, the fluorous initiator is afluorous siloxane or fluorous silane. In one embodiment, the fluorousinitiator is a fluorous siloxane. In one embodiment, the fluorousinitiator is a fluorinated polysiloxane. In one embodiment, the fluorousinitiator is selected from poly(3,3,3-trifluoropropylmethylsiloxane),bis(tridecafluoro-1,1,2,2-tetrahydrooctyl) tetramethyldisiloxane,heptadecafluoro 1,1,2,2-tetrahydrodecyl)dimethylchlorosilane,bis(tridecafluoro-1,1,2,2-tetrahydrooctyldimethylsiloxy)-methylchlorosilane, andbis(heptadecafluorodecyl)tetramethyldisiloxane. In another embodiment,the fluorous initiator is selected frompoly(3,3,3-trifluoropropylmethylsiloxane), bis(tridecafluoro-1,1,2,2-tetrahydrooctyl)tetramethyldisiloxane andbis(heptadecafluorodecyl)-tetramethyldisiloxane. In one embodiment, thefluorous initiator is bis(heptadecafluorodecyl)-tetramethyldisiloxane.

In certain embodiments, a method of preparing a porous semiconductorsubstrate comprises: (1) etching the semiconductor to make a poroussurface and (2) contacting the porous surface with a fluorous initiatorand a photoactive compound. In one embodiment, the fluorous initiatorand photoactive compound is contacted as a mixture to the poroussurface. In yet another embodiment, the fluorous initiator is contactedwith the porous surface before the photoactive compound is contactedwith the porous surface. In yet another embodiment, the photo activecompound is contacted with the porous surface before the fluorousinitiator is contacted with the porous surface.

In certain embodiments, a method of preparing the porous semiconductorsubstrate further comprises the step exposing the substrate to acidicvapor, basic vapor or volatile compounds that specifically react withcertain functional groups. Gas-phase chemical modification of thesubstrate before or after sample deposition occurs in a diffusionalmanner that maintains the addressability and discreteness of the samplesdeposited on the surface. The exposure of the substrate to acidic orbasic vapor was found to enhance detection of the analyte in bothpositive and negative mode ionization. Exposure of the substrate tovolatile reactive reagents in the gas-phase was found to enhance thedetection of classes of compounds having specific functional groups.

In one embodiment, the acidic vapor is selected from TFA, hydrochloricacid and sulfuric acid. In one embodiment, the basic vapor is selectedfrom ammonium hydroxide and ammonium fluoride. In one embodiment, thevolatile compounds are compounds that selectively modify functionalgroups selected from ketones, carboxylic acids, sugars, phosphategroups, thiols and amino groups. In another embodiment, the volatilecompounds selectively modify ketones, carboxylic acids and amino groups.In another embodiment, the volatile compounds are selected fromO-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine, 1,2-phenylenediamine andmethyl isothiocyanate. In yet another embodiment, the acidic vapor, thebasic vapor, or the volatile compounds that react with functional groupsare selected such that their ionization product does not fall within themass range being detected.

In one embodiment, the photoactive adsorbate is a photoactive compoundcontaining a fluorous group. In certain embodiments, the photoactivecompound is an acid which was found to enhance the analysis of theanalyte in positive mode ionization, serving as a proton donor uponirradiation. In certain embodiments, the photo active adsorbatecontaining a fluorous group is selected from(4-Bromophenyl)diphenylsulfonium triflate, (4-Chlorophenyl)diphenylsulfonium triflate, (4-Fluorophenyl)diphenylsulfonium triflate,(4-Iodophenyl) diphenylsulfonium triflate,(4-Methoxyphenyl)diphenylsulfonium triflate, (4-Methylphenyl)diphenylsulfonium triflate, (4-Methylthiophenyl)methyl phenyl sulfoniumtriflate, (4-Phenoxyphenyl) diphenylsulfonium triflate,(4-Phenylthiophenyl)diphenylsulfonium triflate,(4-tert-Butylphenyl)diphenylsulfonium triflate,(tert-Butoxycarbonylmethoxynaphthyl)-diphenylsulfonium triflate,1-Naphthyl diphenylsulfonium triflate,2-(4-Methoxystyryl)-4,6-bis(trichloromethyl)-1,3,5-triazine,Bis(4-tert-butylphenyl)iodonium p-toluenesulfonate,Bis(4-tert-butylphenyl)iodonium perfluoro-1-butanesulfonate,Bis(4-tert-butylphenyl)iodonium triflate,Boc-methoxyphenyldiphenylsulfonium triflate, Diphenyliodoniumhexafluorophosphate, Diphenyliodonium nitrate, Diphenyliodoniump-toluenesulfonate, Diphenyliodonium perfluoro-1-butanesulfonate,Diphenyliodonium triflate, N-Hydroxynaphthalimide triflate,N-Hydroxy-5-norbornene-2,3-dicarboximide perfluoro-1-butanesulfonate,Triarylsulfonium hexafluoroantimonate, Triarylsulfoniumhexafluorophosphate, Triphenylsulfonium perfluoro-1-butanesufonate,Triphenylsulfonium triflate, Tris(4-tert-butylphenyl)sulfoniumperfluoro-1 butanesulfonate, Tris(4-tert-butylphenyl)sulfonium triflateand 5, 10, 15, 20-Tetrakis (pentafluorophenyl) porphyrin. In yet anotherembodiment, the photoactive adsorbate is selected fromtriphenylsulfonium perfluoro-1-butanesulfonate, N-hydroxynaphthalimidetriflate and 5,10, 15, 20-tetrakis(pentafluorophenyl) porphyrin. In yetanother embodiment, the photoactive adsorbate yields an ionizationproduct that does not have an ionized mass that falls within the massrange being detected. In yet another embodiment, the photoactivecompound is a base. The photoactive bases were found to improvenegative-mode ionization detection by serving as activate protonacceptors upon irradiation.

In yet another embodiment, provided herein is a substrate comprising aporous semiconductor, bis(heptadecafluorodecyl)-tetramethyldisiloxaneadsorbed to the semiconductor and a photoactive compound containing afluorous group adsorbed to the semiconductor selected fromperfluoro-1-butanesulfonate, N-hydroxynaphthalimide triflate and 5,10,15,20-tetrakis(pentafluorophenyl) porphyrin.

In yet another embodiment, the substrate is a thermal insulating polymercontaining thermally insulating microwells designed to confine the heatfrom the irradiation beam to the microwell. The thermally insulatingmicrowell can hold a volume of sample and confines heat to themicrowells in which the sample is contained. Thermally confinedmicrowells may be generated using traditional lithographic methods inwhich the substrate surface is coated with thermally insulatingmaterials and etched to form microwells.

In yet another embodiment, the substrate further comprises patternedelectrodes, to which, after sample deposition, an electric potential maybe applied thereby separating complex sample mixtures on the surface bytheir electrophoretic mobility, which can be further enhanced bychanging pH, salt content, or applied voltages/polarity.

In yet another embodiment, provided herein is a kit comprising asubstrate comprising a porous semiconductor, a fluorous initiatoradsorbed to the semiconductor and a photoactive compound adsorbed to thesemiconductor. In another embodiment, the kit further comprises aUV-protective container.

In embodiments, disclosed herein is a desorption/ionization MStechnology platform operable to permit label free analysis of, forexample, small molecule compounds, peptides, proteins, metabolites,biomolecules, cell lysates, whole cells, biofluids and tissues, withhigh sensitivity across a biologically relevant mass range. In oneembodiment, the mass range being detected is from about 70 to about 2000Da. In one embodiment, the mass range being detected is from about 10 toabout 2500 Da. In yet another embodiment, the mass range being detectedis from about 2500 to about 50000 Da.

In certain embodiments, data acquisition is performed using a laserdesorption/ionization (LDI) mass spectrometer. MS instruments includebut are not limited to LDI-TOF, LDI-TOF-TOF, LDI-QTOF, LDI-QQQ,LDI-IMS-TOF, LDI-IMSQTOF, LDI-IMS-QQQ. In embodiments, instrumentsdisclosed herein are capable of analyzing high density libraries orsamples printed on the substrate provided herein. Full m/z spectrum maybe obtained for each sample or each analyte using the instrumentsdisclosed herein. In embodiments, instruments disclosed herein are ableto resolve the masses of analytes that differ by less than 0.1 m/z.Ionization intensity may also be gathered for each analyte using theinstrumentation disclosed herein. In some embodiments, the laser usedemits in the ultraviolet range of the spectrum. In one embodiment, thelaser source is nitrogen or Nd:YAG (frequency-tripled) laser-source.

In embodiments as disclosed herein, significant enhancement in sampleionization, desorption and detection, even of complex mixtures, can beachieved by combining low volume deposition of sample droplets with useof the substrate provided herein which is also designed to enhance theionization and desorption of the samples deposited on the surface. Thishighly focused sample droplet is particularly suited to thethermally-driven ionization process that occurs on the substrate duringionization desorption. Not wishing to be bound by theory, it isbelieved, nevertheless, that upon acoustic deposition of a sampledroplet, the droplet, during flight, partially evaporates before makingcontact with the substrate, and that this rapid evaporation concentratesthe analytes within the drop to metastable concentration levels to forma highly concentrated (with respect to the analyte concentration),focused spot which may have a diameter comparable to the diameter of thelaser used for desorption. Such a highly concentrated spot would bedifficult or impossible to achieve by direct application of the sampleto the surface. It is further believed that because the analyte ishighly focused and concentrated within the circumference of theionization beam, and because the heat intensity is greatest at thecenter of the beam, the number of analytes exposed to the localized areaof heat is maximized, resulting in an increase in the number of analytesthat are desorbed and ionized upon irradiation.

Provided herein, in one embodiment, is a method for non-contactdeposition of samples in volumes ranging from picoliter(s) tonanoliter(s) onto the substrate. The deposition may be a continuoussurface coating or have micron scale separation. In certain embodiments,an acoustic dispenser is used to deliver samples in the single nanoliter(nL) to high picoliter (pL) range. In certain embodiments, the sampledroplet size is no larger than the width of the ionization beam. Wherethe ionization beam is a laser beam having a diameter of approximately40 microns, the sample droplet may be dispensed in the low nanoliterrange to generate a droplet size comparable to the diameter of thelaser. In certain embodiments, the laser diameter is from about 7 μm toabout 10 μm, in which case the sample droplet is dispensed at a volumeof from about 0.5 picoliters to about 1 nanoliter.

In embodiments, non-contact deposition is used, wherein a manner ofsample deposition is employed where no foreign surface other than thesurface of the well or container holding the sample touches the sampleduring sample deposition. For example, no foreign surface such as a tip,pin or capillary device is used to transfer the sample in a non-contactsample deposition. In one embodiment, non-contact deposition is achievedusing an acoustic liquid dispenser. Examples of acoustic liquiddispensers are ATS-100 by EDC Biosystems and the Echo series of liquidhandlers by Labcyte, Inc.

In some embodiments, a sample volume of about 1 μL or less is applied tothe substrate using a low-volume pipette or acoustic deposition. In someembodiments, a sample volume of less than about 1 μL is applied to thesubstrate. In some embodiments, a sample volume of about 0.1 to about 10nL is applied to the substrate using acoustic deposition.

Provided herein are embodiments of a method of detecting an analyte in asample by desorption ionization mass spectrometry, comprising the stepsof (1) depositing a sample having a volume in the picoliter to nanoliterrange on a substrate, (2) delivering radiation to said sample to causedesorption and ionization of said sample and (3) detecting themass-to-charge ratio of the ionized analyte. In some embodiments, thesample deposition step is a noncontact deposition step. In someembodiments, the sample deposition step is performed using an acousticliquid dispenser. In some embodiments, the sample deposition step isperformed using an acoustic liquid dispenser and the volume deposited isfrom about 1 nanoliter to about 5 nanoliters. Also provided herein areembodiments of a method of detecting an analyte in a sample bydesorption ionization mass spectrometry, comprising the steps of (1)depositing a sample having a volume from about 1 nanoliter to about 5nanoliters onto a substrate using non-contact deposition, (2) deliveringradiation to said sample to cause desorption and ionization of saidsample and (3) detecting the mass-to-charge ratio of the ionizedanalyte.

Also provided herein are embodiments of a method of detecting an analytein a sample by desorption ionization mass spectrometry, comprising thesteps of (1) depositing a droplet of sample, (2) delivering radiation tosaid sample to cause desorption and ionization of said sample, and (3)detecting the mass-to-charge ratio of the ionized analyte. In someembodiments, the sample has a volume in the range of about 1 to about 5nanoliters. In some embodiments, the radiation is from a laser source.In some embodiments, the laser source is an ultraviolet pulse lasersource. In some embodiments, the ultraviolet pulse laser is a 337 nmpulsed nitrogen laser. In some embodiments, the laser source is a Nd:YAG(neodymium-doped yttrium aluminium garnet) laser source. In yet anotherembodiment, the laser source is a Nd:YAG laser source. In someembodiments, the radiation is from an ion beam source. In someembodiments, the ion beam is comprised of ions selected from the groupconsisting of Bi₃+, Bi+, Au+ and Ga+.

One of the challenges of working with label-free samples or librariesprinted on surfaces (such as the substrates provided herein) is thatsuch label-free samples or libraries are usually not detectable, usingoptical methods or otherwise, and therefore locating or identifyingsignificant features and aligning features between different substratesfor comparison purposes is extremely difficult. One analytical approachto circumvent the problem is to use clustering and/or dimensionreduction techniques to elucidate significant features or attributesfrom the collective data rather than identifying or analyzing discretedata points. Provided herein is a method of analyzing the spectral dataand elucidating significant features or distinct attributes from thedata using an algorithm comprising the following steps: (1) gatheringall spectral data obtained from a surface or multiple surfaces, whichare treated as independent measurements (2) identifying valid peaks inall spectra (using well-established peak detection methodologies such aswavelet-based spectral decomposition) and aligning them to a common axisto correct for slight peak shifts that may occur due to differencesbetween substrates or differences between different areas of the samesubstrate, (3) normalizing aligned peaks to correct for overall signalintensity variation between individual spectra (for example using aglobal median intensity, total intensity, mean intensity, local medianintensity or other applicable measures) (4) performing clusteringanalysis using one or more well established clustering and/or dimensionreduction methods (for example, k-means clustering, singular valuedecomposition, multidimensional scaling, principle components analysis,self-organizing maps, learning algorithms or others) and (5) identifyinga significant feature or distinct attribute (such feature or attributemay be, for example, compounds within a library with differing activityand/or mode of action, different cellular phenotypes within a library ofcells or a cell-based screen, different organism phenotypes within alibrary of whole-organisms or an organism-based screen, differentregions of cellular activity within a tissue on a surface, and/orenzymes with differing activity). The method may further comprise thestep of mapping the statistically significant sets of features orattributes back to one or more regions on the original surface thatcontained the samples or libraries.

The foregoing method is useful in metabolomic or proteomic studies, inwhich the metabolite profile or the protein profile of a cell, cellcompartment, tissue or organism may be analyzed to generate a profile orfingerprint, of a disease state, for example, where the sample isobtained from a disease-specific cell line, diseased tissue or diseasedorganism. For example, a metabolite profile or fingerprint from adiseased specimen may be compared to a metabolite profile of a healthyspecimen. Any detected increase or decrease in activity of a particularmetabolic pathway or pathways could help identify the biologicalprocesses underlying a disease. Similarly, protein profiling of adisease state could lead to the identification of useful biomarkers forthe disease. The metabolic or protein profile that is generated, incombination with the data analysis method disclosed above, may also beused to diagnose or classify diseases. Such a profile could be used toclassify or define a disease at the molecular level and may permit earlydiagnosis, early treatment and personalized treatment of the diseasebased upon the profile.

The substrates, methods and kits provided herein may be used as tools inbiomedical and biomolecular research. The substrates, methods and kitsprovided herein may be used to perform compound library analysis,enzymatic assay, cell based assay, drug distribution study, tissueprofiling, tissue imaging, metabolic profiling studies, proteinprofiling studies, biofluid analysis, drug metabolite analysis and drugtesting. The substrates, methods and kits provided herein haveindustrial applications, including the research and development ofindustrial enzymes and bacteria.

EXAMPLES Example 1

The miniaturization of library samples was accomplished by acousticallyapplying 1 nL droplets from cellulose assay samples stored in multi-wellmicrotiter plates to a MS substrate. FIG. 7 is an optical microscopicimage of a portion of the resulting acoustically printed array. Heresamples that were 1 mm apart in 3 mm high wells are transformed into anMS array that is 0.18 mm apart and 0.5 mm high (the thickness of the MSsubstrate), a more than 30-fold densification.

Example 2

An alpha-amylase enzyme was incubated with two starch sources, potatoamylose and corn amylopectin diluted in 20 mM sodium acetate buffer in astandard 96-well microtiter plate. The enzyme reaction was run for 90minutes at 95 C in an incubator. After 90 minutes, the enzyme reactionwas quenched with methanol 1:1 (vol:vol).

6 μL of quenched reaction mixture was transferred from the 96-well plateto a 1536-well film-bottom microtiter plate. The 1536-well plate wasmounted to the source stage of an EDC ATS-100 acoustic liquid transfersystem. A silicon mass spectrometry chip was mounted to a SBS formatcarrier frame, compatible with a Bruker laser desorption/ionization massspectrometer, and containing fiducial marks for alignment, then mountedactive-side down in the target stage of the EDC ATS-100 such that theactive side of the mass spectrometry chip was 2 mm above the 1536-wellsource plate on the source stage. A plurality of samples was printedacoustically from the source plate onto the mass spectrometry chip as arectangular array of 20 nL drops with a center-to-center spot pitch of400 microns and all positions relative to the fiducial marks.

The printed mass spectrometry chip attached to the SBS carrier frame wascovered with a plastic protective cover to prevent physical damage. Anon-leaching, dust-free desiccant cartridge was attached to the bottomof the carrier frame with an attachment clip on the back of the carrier.A Mylar sleeve was placed over the entire apparatus and heat sealed atone end. The open end of the Mylar sleeve was placed in a vacuum sealerto evacuate the interior of the Mylar sleeve, then heat sealed. Theentire apparatus was then placed in an envelope and shipped from SanDiego, Calif. to Fremont, Calif. via air cargo.

Mass spectrometry data acquisition was performed at the remote analysisfacility. The SBS carrier frame containing the printed sample array massspectrometry chip was removed from the Mylar sleeve and desiccantcartridge removed from the attachment clip and it was found that theshipping container had preserved the physical integrity of the arrayduring transport. Analysis of the chemical composition at the remotefacility was performed using a Bruker laser desorption/ionization massspectrometer. The fiducial marks enabled registration of the sample. Thefirst position of the sample array was taught to the mass spectrometertarget stage relative to the same fiducial marks used for acousticsample deposition, enabling data acquisition to be performed on aper-spot basis where each mass spectrum contained a row/col ID in thedata file header. The position of each acquired mass spectrum wasrecorded and correlated to the sample ID at each specific location. Datawas made available in digital format for facile remote access by thecustomer via the internet. FIG. 8 shows an enzyme assay mass spectrumacquired from a specific position on the spatially encoded amylaseenzyme array after storage/transport.

Example 3

96 urine samples, some of which contain illicit drug metabolites, weremixed 1:1 (vol:vol) with acetonitrile containing 0.1% trifluoroaceticacid in a 96-well plate with each extraction mixture in a separate well.

6 μL of the extraction mixture was transferred to a 1536-wellfilm-bottom microtiter plate. The 1536-well plate was mounted to thesource stage of an EDC ATS-100 acoustic liquid transfer system. Asilicon mass spectrometry chip was mounted to a SBS format carrier framecompatible with a Bruker laser desorption/ionization mass spectrometercontaining fiducial marks for alignment, then mounted active-side downin the target stage of the EDC ATS-100 such that the active side of themass spectrometry chip was 2 mm above the 1536-well source plate on thesource stage. 96 samples were printed acoustically from the source plateonto the mass spectrometry chip as a rectangular array of 10 nL dropswith a center-to-center spot pitch of 400 microns and all positionsrecorded relative to the fiducial marks.

The printed mass spectrometry chip was packaged as described in Example2 and stored for four days at −20 C before analysis. The entireapparatus was then placed in an envelope and shipped from San Diego,Calif. to Fremont, Calif. via ground transportation.

The SBS carrier frame containing the printed sample array massspectrometry chip was removed from the Mylar sleeve and desiccantcartridge removed from the attachment clip. The entire frame wasinserted into a Bruker laser desorption/ionization mass spectrometer.The first position of the sample array was taught to the massspectrometer target stage relative to the same fiducial marks used foracoustic sample deposition. Data acquisition was performed on a per-spotbasis where each mass spectrum contained a row/col ID in the data fileheader. Data was delivered to the customer via a portable USB memorystick.

FIG. 9 shows a spatially encoded transportable urine sample analysiswith mass spectrometry.

FIG. 10 shows a mass spectrum obtained from a specific urine sample onthe spatially encoded array after storage/shipping.

Example 4

Human liver microsomes (HLMs) expressing the CYP2D6 enzyme wereincubated with 10 μM dextromethorphan for 7 minutes at 37 C in 25 mMpotassium phosphate buffer containing 1 mM NADPH, 2 mM MgCl and 700 nMof isotope labeled dextrorphan internal standard used for calibrationand quantitation. After 7 minutes, the reaction was quenched withacetonitrile 3:1 (vol:vol).

6 μL of the quenched reaction mixture was transferred to a 1536-wellfilm-bottom microtiter plate and centrifuged for 5 minutes at 200×g. The1536-well plate was mounted to the source stage of an EDC ATS-100acoustic liquid transfer system. A Bis-F17 coated NIMS mass spectrometrychip was mounted to a SBS format carrier frame compatible with a Brukerlaser desorption/ionization mass spectrometer containing fiducial marksfor alignment, then mounted active-side down in the target stage of theEDC ATS-100 such that the active side of the mass spectrometry chip was2 mm above the 1536-well source plate on the source stage. 96 sampleswere printed acoustically from the source plate onto the massspectrometry chip as a rectangular array of 15 nL drops with acenter-to-center spot pitch of 400 microns and all positions relative tothe fiducial marks. In this manner the sample was printed as aminiaturized spatially encoded sample array on a mass spectrometrysurface after which it was stability-packaged as described in Example 2and stored for two weeks at −20 C before analysis. Thestability-packaged sample arrays were placed in an envelope and shippedvia air from San Diego, Calif. to Hartford, Conn. for mass spectrometrydata acquisition. Mass spectrometry data acquisition was performed suchthat the location of each acquired mass spectrum was recorded andcorrelated with sample ID on the spatially encoded sample array. Datawas delivered to the customer remotely via the internet. FIG. 11 shows amass spectrum obtained from a specific drug toxicity assay sample on thespatially encoded array after storage/shipping, showing the correctexact mass peak 480 for this compound and the internal standard,2H3-dextrorphan 490.

Example 5

A 1536-well plate of drug candidate molecules dissolved in DMSO anddiluted to 40 μM concentration was printed using an EDC ATS-100 acoustictransfer system onto a mass spectrometry surface as a spatially encodedarray according to the method described in Example 2. 96 samples wereprinted acoustically from the source plate onto the mass spectrometrychip as a rectangular array of 2 nL drops with a center-to-center spotpitch of 400 microns and all positions relative to the fiducial marks.The mass spectrometry surface stability-packaged as described in Example2 and preserved for 3 days. The stability-packaged array was shippedfrom San Diego, Calif. to Fremont, Calif. by air cargo for mass spectrumdata acquisition. The SBS carrier frame containing the printed samplearray mass spectrometry chip was removed from the Mylar sleeve anddesiccant cartridge removed from the attachment clip. The entire framewas inserted into a Bruker laser desorption/ionization massspectrometer. The first position of the sample array was taught to themass spectrometer target stage relative to the same fiducial marks usedfor acoustic sample deposition. Tandem mass spectrometry dataacquisition was performed on a per-spot basis where each mass spectrumcontained a row/col ID in the data file header. Parent and tandem massspectra were acquired from each sample such that the spatial location ofthe parent and tandem mass spectrum were recorded and correlated back toa specific sample ID. Data was delivered to the customer remotely viathe internet. FIG. 12 shows a tandem mass spectrum obtained from aspecific drug molecule sample on the spatially encoded array afterstorage/shipping.

Example 6

Integration of acoustic printing and MS analysis registration systems atthe time of printing. Here sample registration on an MS array wasmaintained between the point of printing and the point of massspectrometry readout so as to facilitate tracking of MS samplepositions, with three or more fiducial markers being used for alignment.MS substrates were mounted in a machined steel frame designed to hold upto two chips. The frame has SBS dimensions for robotic handlingcompatibility. Fiducial points were positioned at each of the fourcorners of the frame, and each chip mounting position has an indexedcorner that the upper left corner of the mass spectrometry chip isplaced tightly against. The four fiducial points on the frame are usedas spatial reference points for acoustic array printing on the massspectrometry chips mounted to the frame. For mass spectrometry readout,the frame is mounted to a target plate compatible with the particularmass spectrometer being used. The same four fiducial points on the frameare then used to program the X-Y axis of the mass spectrometer so thatthe X-Y origin matches that used during acoustic printing using an EDCATS-100. At the time of printing a GAL (Genepix Array List) file isgenerated, which is not a standard output for acoustic transfer systemsbut was adapted for use. A MS plate map was computed from the GAL fileand written to a file in a format compatible with the mass spectrometerstage software. This plate map file defined the center of each arrayspot relative to the X-Y axis origin, and may include the diameter ofeach spot. By calibrating the mass spectrometer stage to the same X-Yaxis origin as was used during printing, and subsequently defining eacharray spot center relative to that same X-Y axis origin, spotregistration was maintained throughout printing, transport, storage, andacquisition. The tracking identifier of each spot was also stored in theGAL file at the time of printing, and also recorded in the generated MSplate map file. As a result, the desired outcome was achieved: eachsample spot spectrum was found to contain the 1) location (x, ycoordinate) on the array and 2) the unique sample ID, and 3) the rawmass spectrum contained within the spot spectrum metadata file generatedby the MS instrument at the time of acquisition.

Example 7

Samples are applied by acoustic printing to a sample-accepting surfaceof an MS substrate. Because acoustic printing is affected by changes inthe speed of sound through the sample media, an initial calibration stepwas performed when printing a new sample composition. (This calibrationcan be utilized during future acoustic printing of the same/similarsample composition.) The calibration process was performed by startingwith low acoustic emission energy and increasing that energy step-wiseto high acoustic emission energy. At each emission energy, the ejecteddroplet volume was calculated by emitting an echo wave that does noteject a droplet but instead reflects off of the sample meniscus. Fromthe time between echo wave emission and reflection (the acoustictransducer also acts as a receiver), given the sample reservoir/wellgeometry, the ejected droplet volume was calculated. Finally thecalibration curve is generated by plotting the acoustic wave emissionenergy vs. the ejected droplet volume.

Once the calibration curve for a specific sample composition has beengenerated, the curve is then used to acoustically eject a specificvolume of 1 nL of sample onto the mass spectrometry chip to produce ahigh-density array consisting of nanoliters/picoliters of a plurality ofsamples. A sample source plate, typically a 96, 384, or 1536-wellfilm-bottom microtiter plate, was mounted to the source stage of an EDCATS-100 acoustic liquid transfer system with the top (open side ofwells) of the plate facing up. The mass spectrometry chip is mounted toan SBS format carrier frame, and the mounted chip is then mounted in thetarget stage of the EDC ATS-100 above the sample source plate such thatthe active surface of the chip was facing down towards the top of thesource plate, with the active surface approximately 2 mm above thesource plate. This second stage to which the mass spectrometry chip ismounted moves independent from the sample source plate stage. Theacoustic printer was then programmed to position a specific source wellover the acoustic horn, and in parallel the chip stage positions themass spectrometry chip over the selected source well such that thedesired array spot placement position was centered over the opening ofthe source well. Then the acoustic horn was programmed to emit anacoustic pulse to eject a droplet of typically 1 nanoliter from thesource well onto the mass spectrometry chip. If more material wasrequired on the array spot, additional droplets are ejected withoutmoving the source plate or the mass spectrometry chip (replicate-on-dropprinting). Additionally, other samples/reagents could have been printedfrom a different source well to the same array spot to performchemistry, add internal standards, or enhance detection.

This process was repeated for a plurality of samples to produce ahigh-density array of many samples on a mass spectrometry chip resultingin array densities of 100-800 micron center-to-center spot pitch.

Example 8

N-Desethylamodiaquine was diluted to 1 mM in 20 mM potassium phosphatebuffer with 700 nM D(5)-N-desethylamodiaquine internal standard, thenmixed 1:1 (vol:vol) with a solution of 97% water 3% acetonitrile and0.1% trifluoroacetic acid. The resulting mixture was transferred to a1536-well microtiter plate with a film-bottom compatible with acousticsample printing. An array of 2 nL sample droplets was transferred to anMS substrate and allowed to dry. The MS substrate was analyzed in SanDiego, Calif. on a Bruker Autoflex mass spectrometer within 4 hours ofsample deposition. The resulting spectrum, shown in FIG. 15A, includespeaks corresponding to N-Desethylamodiaquine 500 and the internalstandard 505. The MS substrate was then packaged in an environmentallycontrolled chamber consisting of a plastic holder to hold the substratein place and to protect it from physical contact and a dehydratingcartridge just outside the plastic holder. These were then placed in amylar film bag that was then evacuated with vacuum and heat sealed witha hot-strip. This sealed apparatus was shipped via ground transport toFremont, Calif. Upon arrival in Fremont, Calif., the MS substrate wasremoved from the packaging for subsequent analysis. A second massspectrum was acquired from the same sample array using a BrukerUltraflex mass spectrometer. The resulting spectrum is shown in FIG. 15Band again includes peaks corresponding to N-Desethylamodiaquine 510 andthe internal standard 515.

Example 9

An assay screen of starch digestion enzymes was performed. At a customersite, the reaction mixtures, including internal standards, wereacoustically printed using a EDC Biosystems ATS acoustic printer, in anarray of 800 um spot pitches, onto an electrochemically etched (usinghydrofluoric acid) porous silicon 75 mm by 25 mm mass spectrometry chipcoated with a thin film, secured in an SBS format chip holder forcompatibility with the instrument. More than 6000 spots were printed,including at least 4 replicates of each of more than 1500 samples. Thesamples included more than 100 unique enzyme variants under 3 differentconditions and time points of 0, 1, 12, 24, and 48 hours. Each spot wasprinted 4 times, spot on spot, at 5 nL to provide a sample volume of 20nL per spot. A positional file was also produced at the customer site torelate spot location to a sample ID for each spot. The positional fileincluded array identification information, barcode, and layoutinformation including number of blocks, first spot offset position,number of rows and columns, and spot center to center distance (pitch);as well as spot specific information including, for each spot, block,row, column, sample identifier, and three experiment descriptionparameters. Samples were blinded by using arbitrarily assigned numbersas sample identifiers, with the identity of the enzyme variants in eachsample remaining under the exclusive control of the customer.

The chip containing the array of samples was then packaged into a chipcase (Electron Microscopy Services Plastic Single Slide MailerCat#71552-01). The chip case, containing the chip, was then placed intoa mylar bag and the mylar bag was evacuated and sealed to hold a vacuumstate within the bag. The bag, containing the chip and chip case, werethen transported from the customer site approximately 2 miles to anoff-site mass spectrometry facility. The chip was then un-packaged,placed onto a mass spectrometry chip holder in the same orientation asused during printing, loaded into a mass spectrometer. The positionalfile was electronically transported to the mass spectrometry site. Thepositional file was converted to a mass spectrometry positional file andloaded onto the mass spectrometer. The chip was positioned manually tothe first spot to ensure correct register. The mass spectrometer thencollected the data from the samples on the chip assigning theappropriate sample ID to the mass spectra according to the positionalfile information. These results were analyzed and presented to thecustomer. FIG. 16 shows a sample spectrum for one of the blindedsamples, with a peak 520 corresponding to the analyte and demonstratingdetection of the internal standards at m/z values of approximately 199(tacrine 525), 1046 (angiotensin II 530), and 1060 (bradykinin 535).

Example 10

(In Examples 10 through 14, mass spectrometry was performed on AppliedBiosystems 5800 TOF-TOF. Acoustic dispensation was performed usingATS-100 from EDC Biosystems.)

Substrates were prepared as follows: P-type silicon wafers were cut tothe desired dimensions and cleaned with ethanol followed by methanol anddried with a nitrogen gas stream. The silicon substrate was etched byacidic electrochemical etching for 30 minutes in a solution of 25%hydrofluoric acid in ethanol and a constant current of 300 mA. Afteretching, the silicon substrate was rinsed with water, then methanol anddried with a nitrogen gas stream and baked for 15 minutes at 100° C. Athin layer of bis(heptadecafluorodecyl)-tetramethyldisiloxane initiatorsolution containing one or more of the photoactive additivesN-hydroxynaphthalimide triflate (500 μM), triphenylsulfoniumperfluoro-1-butanesufonate (500 μM) or5,10,15,20-tetrakis(pentafluorophenyl) porphyrin (25 mM) were then addedto the substrate for one hour, after which excess initiator was removedwith a high flow stream of nitrogen gas. Either before or after sampledeposition on the substrate, the surface was further modified with gasphase treatment using one of the following reagents: trifluoroaceticacid vapor, hydrochloric acid vapor, ammonium hydroxide vapor, ammoniumfluoride vapor, 0-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine vapor,1,2-phenylenediamine vapor, or methyl isothiocyanate vapor. Thesubstrate can be stored for many weeks in a dry environment/chamberwithout loss of performance.

FIG. 17 is a spectral comparison of blood sample detected on a substratewith (bottom spectra) and without (top spectra) photoacid treatment ofthe substrate. The photoacid treatment significantly enhances the numberof peaks detected.

FIG. 18 is tissue imaging of a mouse brain slice with and withoutphotoacid treatment. The top half of the brain slice was treated withphotoacid and shows significant enhancement of signal compared to thebottom half of the sample that was not treated with photoacid.

Example 11

High through put analytical characterization of chemical libraries wascarried out. 1280 compounds from a small molecule library were depositedon the substrate described in Example 10. The compounds had an initialconcentration of 10 mM in ˜70% DMSO/water. The compounds were dilutedthirty-fold from a 384-well acoustic source plate to a 1536 wellacoustic source plate in 50% DMSO/water. The plate was centrifuged at1500 rpm for 3 minutes to remove air bubbles and seat the liquid on thebottom of each well. The acoustic source plate was then placed in theacoustic dispenser and about 0.8-1 nL of each well was spotted onto thesubstrate in a direct one-to-one transfer maintaining the same welllayout.

In a similar manner, a 100-fold dilution was tested as well as 200-folddilution. In the 200 fold dilution test we transferred 40 nL of the 10nM solution to a 1536 well acoustic source plate. 7.960 μL of a 50%DMSO/water 1% TFA solution was added to each well and the plate wastreated in a similar manner as above and spotted onto our proprietarychip in a direct one to one transfer keeping the same well layout.

Data acquisition was performed using an Applied Biosystems 5800 TOF-TOFlaser-desorption/ionization (LDI) mass spectrometer equipped with anNd:YAG laser fired at 400 Hz and 1500-4000 laser power.

FIG. 19 shows the intensity map of the spectral data for each compoundanalyzed at 30-fold dilution and example spectra that are generated foreach compound screened. The generally consistent intensity of the peaksindicates consistency of ionization of compounds.

Example 12

Metabolic profiling of drug activity in single cells was performed.Burkitt's lymphoma-derived cells (Raji cells) were either untreated ortreated with 50 μM rapamycin for 1 hour at 37° C. A sparse population ofthese cells were deposited with a pipette on the substrate at a volumeof 0.50 μL with less than 100 cells contained within the spot. Thesurface was analyzed with scanning laser desorption mass spectrometry toproduce a spectrum at each pixel scanned.

FIG. 20 shows the spectra obtained from a treated sample overlaid withspectra obtained from an untreated sample. The drug (m/z 936.54 (M+H⁺)was detected in treated cells but not in the untreated sample.

Example 13

High-throughput liver metabolite profiling of genetic knockout mice wasperformed. Livers dissected from PNPLA3-knockout and wild type mice wereeach desiccated then ground to a fine homogenized powder. I mg of thepowder was dissolved in 40% methanol, 10% chloroform and 50% water andsonicated for 5 minutes. The samples were then centrifuged at 2,000 gand the supernatant collected. 4 μL of each liver extract sample wastransferred from the spiked source wells to an acoustic 1536 well platein a 6×6 square. Yellow food coloring was prepared (300 μL to 25 mL 75%methanol/water) and added to the acoustic source plate as an index alongone outlying row and one outlying column to mark the orientation of thesubstrate. The silicon substrate described in Example 10 was spotted in5 nL volumes in an addressable format, in 12 multiplets to make an 18×24spot grid with an outer edge of dye along the right side and on thebottom for orientation in the mass spectrometer instrument. The sampleswere analyzed with both positive and negative mode mass spectrometryacross the 100-1000 m/z range. Using the data analysis algorithmdescribed herein, the raw mass spectrometry data was processed toidentify significant peaks and their location on the substrate—whichcorresponds to either a wild-type or knockout liver extract.

FIG. 21 shows the metabolic profile spectrum of a PNPLA3-knockout mouseliver extract overlaid with the metabolic profile spectrum from the wildtype liver extract showing significant metabolic profile differences.

Example 14

Chemical library screening using whole-organism zebrafish was performed.96 wells containing one, two or three zebrafish at the Prim-15 stage ofdevelopment were treated with compounds from a small molecule library at10 μM concentration for 1 hour. After 1 hour, growth media was drawn offand 150 μL of methanol was added to the wells and the samples were flashfrozen and stored at −80° C. Frozen samples were sonicated in an icewater bath for 15 minutes and further homogenized with repeatedpipetting cycles within the wells and the well plates were spun down.

4 μL of the zebrafish sample extracts in 90% methanol/chloroform werecarefully decanted off the top of the well and placed in a 384 wellacoustic source plate in a 10 by 10 grid. The outer columns and wellswere filled with a dilute food coloring to form a 12 by 12 grid. Theplate was then “stamped” into a 1536 well plate with 4 replicates usinga Janus 384 well head liquid handler. The plate was centrifuged at 1500rpm for 3 minutes to remove air bubbles and seat the liquid on thebottom of each well.

The acoustic source plate was then placed in the acoustic dispenser andapproximately 1 to about 5 nL of each well was spotted onto thesubstrate described in Example 10 as a 24 by 24 grid with a spot area ofapproximately 0.008 mm². Spectra from the array spots were acquiredusing positive and negative mode mass spectrometry and raw data analysiswas performed using the data analysis algorithm described herein.

FIG. 22 shows the metabolic profile of a zebrafish sample treated with alibrary compound. The sample was analyzed for detection in the 100 to1,000 m/z range, and the spectrum was obtained in positive ionizationmode. Another spectrum, not shown, was obtained in negative ionizationmode.

FIG. 23 shows an intensity map of a selected metabolite, phosphocoline,detected in the whole organism compound library screen in zebrafish.Each sample represents the phosphocoline detected in a zebrafishorganism incubated with a compound from a library.

FIG. 24 shows the cluster analysis of the zebrafish array in which thespectral profile obtained from the array is clustered using thealgorithm described herein. Distinct groups of cellular or metabolicactivity could be distinguished from background or background activity.

CONCLUDING MATTER

Aspects and/or embodiments of the disclosure hereof include, withoutlimitation and by way of example, any of the following, singly or in anyoperable combination:

In an aspect, there is provided a stability-packaged mass spectrometric(MS) array including a MS substrate having a plurality of MS samplesapplied thereon, an enclosure surrounding the MS samples, anenvironmental control system, and a protective barrier isolating the MSsamples from physical contact.

In an aspect, a stability-packaged MS array includes a miniaturizationof a sample library or subset thereof, and the packaging volume occupiedby the MS samples and MS substrate is less than about 1/1000 thepackaging volume of the sample library or subset thereof from which theMS samples were miniaturized.

In an aspect, a stability-packaged MS array includes MS samples locatedin a trackable position.

In an aspect, a stability-packaged MS array includes a sealed enclosure.

In an aspect, a stability-packaged MS array includes an enclosure thatis fully or partially evacuated.

In an aspect, a stability-packaged MS array includes at least one of theMS samples immersed in a non-interactive gas or fluid, which may be agas selected from: ultra-high purity (UHP) nitrogen, argon, or an inertgas.

In an aspect, a packaged MS substrate includes an environmental controlsystem disposed and configured to condition an aspect of the environmentproximate to the MS samples selected from the temperature, the humidity,the gas or fluid composition, or the particulate content, and mayinclude a desiccant or humidity control device and/or acontaminant-suppressant composition or package and indicate adverseconditions during shipping.

In an aspect, a stability-packaged MS array includes at least one MSsample present in a quantity insufficient for NMR determination of theprecise chemical structure of the MS sample.

In an aspect, a stability-packaged MS array includes MS samples disposedon an MS substrate at a center-to-center distance of less than about 500microns, and/or at a volume less than about 500 nL.

In an aspect, a stability-packaged MS array includes at least one MSsample disposed on the MS substrate at an analyte concentration of lessthan about 10 mg/mL.

In an aspect, a stability-packaged MS array includes at least one MSsample disposed on the MS substrate at an analyte concentration of lessthan about 1 ng/mL.

In an aspect, a stability-packaged MS array includes at least one MSsample disposed on the MS substrate at an analyte concentration of lessthan about 1 fg/mL.

In an aspect, a stability-packaged MS array includes at least one MSsample including analyte molecules having mass less than about 3,000Daltons.

In an aspect, a stability-packaged MS array includes at least one MSsample including analyte molecules having mass less than about 30,000Daltons.

In an aspect, a stability-packaged MS array includes at least one MSsample including analyte molecules having mass less than about 300,000Daltons.

In an aspect, a stability-packaged MS array includes at least one MSsample including molecules conforming to Lipinski's Rule of Five.

In an aspect, a stability-packaged MS array includes at least one MSsample including an analyte selected from: animal metabolites, humanmetabolites, microbial metabolites, plant metabolites, or drugmetabolites, and/or derived from a source selected from: urine, blood,cerebrospinal fluid, or tissue.

In an aspect, a stability-packaged MS array includes MS samples that arenot captured or covalently bound to the MS substrate.

In an aspect, a stability-packaged MS array includes MS samples that areblinded and/or present in quantities below the threshold for practicableNMR determination of structure.

In an aspect, a MS substrate is mounted in a mounting frame.

In an aspect, a MS substrate or mounting frame includes a fiducial mark.

In an aspect, a MS substrate or a mounting frame in which it is mountedhas dimensions compatible with a standard SBS footprint or a standardmicroscope slide footprint.

In an aspect, a stability-packaged MS array includes a quality controlstandard.

In an aspect, a MS substrate includes a sample-accepting surfaceselected from: a conductive surface, a stainless steel surface, asemiconductor surface, a silicon surface, an etched silicon surface, aliquid coated silicon surface, a glass surface, a plastic surface, or agold surface.

In an aspect, a stability-packaged MS array includes a barrierconfigured and disposed to protect the MS samples thereon from exposureto light.

In an aspect, there is provided a method of packaging a MS substratehaving a plurality of MS samples applied thereto, the method includingenclosing the MS samples in an enclosure, establishing a substantiallywater-vapor-free, particulate-free environment proximate to the MSsamples, and protecting the MS samples from physical contact.

In an aspect, a method includes packaging a MS array according to thedisclosure hereof and dispatching the packaged MS array for offsitetransport.

In an aspect, a method includes receiving information including a MScharacteristic of at least one of the MS samples determined by a MSanalysis performed at an offsite location.

In an aspect, a method includes packaging an MS array in an enclosureand fully or partially evacuating the enclosure and/or disposing aninert gas or fluid within the enclosure and/or disposing a desiccant indiffusive communication with a gas or fluid proximate to the MS sample.

In an aspect, there is provided a method of using a stability-packagedMS array which may include any one or more of: dispatching thestability-packaged MS array for offsite transport; receiving thestability-packaged MS array at a location offsite from the location atwhich MS samples were applied to the MS array; removing the MS arrayfrom its packaging; using the stability-packaged MS array for performingoffsite MS analysis; using the stability-packaged MS array for engagingin the business of performing offsite MS analysis for customers;recording data or results of offsite MS analysis performed on the MSarray; communicating data or results of offsite MS analysis performed onthe MS array; receiving data or results from offsite MS analysisperformed on the MS array; and/or analyzing data or results from offsiteMS analysis performed on the MS array.

In an aspect, there is provided a data management system for offsite MSanalysis including a plurality of MS data records each including atracking identifier and an MS characteristic determined by offsite MSanalysis of an MS sample with which the tracking identifier isassociated, and a data store in which the plurality of MS data recordsare stored and operable for retrieving data stored therein associatedwith a tracking identifier.

In an aspect, a data management system includes a user interfacedisposed and configured to obtain input of a query including a trackingidentifier, transmit the tracking identifier to the data store, andreceive and communicate to a user data retrieved from the data storeassociated with the tracking identifier.

In an aspect, a data management system includes a data store selectedfrom: a relational database, an object-oriented database or datastructure, a no-SQL database, a cloud-based data storage utility, an XMLor SGML-based data structure, a JSON data structure, a flat file; or aspreadsheet file.

In an aspect, a data management system includes MS data records in aformat operable for merging with a data store of a customer entitled tothe results of an offsite MS analysis.

In an aspect, there is provided a non-transitory machine-readable mediumhaving data written thereon including a plurality of MS data records,each MS data record including a tracking identifier and an MScharacteristic determined by offsite MS analysis of an MS sample withwhich the tracking identifier is associated.

In an aspect, a non-transitory machine-readable medium has data writtenthereon in a format operable for merging with a data store of a customerentitled to the results of an offsite MS analysis.

In an aspect, a method includes any or all of: at a loading site,preparing a MS array by applying MS samples to trackable loci on a MSsubstrate by non-contact deposition, associating with the trackable locitracking identifiers corresponding to the MS samples applied thereto,packaging and transporting the MS array, performing at an MS analysissite that is offsite from the loading site a MS measurement to determinea MS characteristic of at least one of the MS samples, and, in a record,associating the MS characteristic of the at least one MS sample with thetracking identifier corresponding to the at least one MS sample.

In an aspect, a method includes protecting MS samples applied to an MSarray against alteration.

In an aspect, protecting MS samples against alteration may includeprotecting the MS samples against contaminants, moisture, particulates,and/or changes in hydration; fully or partially enclosing the MS sampleswithin an enclosure that is fully or partially impervious to a substanceselected from: a particulate, a gas, a vapor, or an aerosol; packagingthe MS array in a package including a physical barrier disposed toestablish a gap between the MS samples and the physical barrier; and/orpackaging the MS array in a package including an environmental controlsystem.

In an aspect, applying a plurality of MS samples to trackable loci on aMS substrate includes a method of application selected from: non-contactdeposition, acoustic deposition, contact printing, piezo printing,pipette printing, or ink jet printing.

In an aspect, a method includes measuring or observing a quality controlproperty of the MS substrate or an MS sample thereon.

In an aspect, a method includes analysis of MS samples obtained from asample library where the library is not accessible at the MS analysissite.

In an aspect, a method includes MS measurement performed using an MSinstrument that is not accessible at the sample loading site.

In an aspect, a stability-packaged MS array is transported via atransport modality selected from: common carrier transport, transport inthe U.S. mail, transport by a freight forwarder, transport by a packageexpress carrier, transport by a courier, transport by aircraft, ortransport by a motor vehicle.

In an aspect, there is provided a system for offsite MS analysisincluding a MS substrate having a plurality of MS samples appliedthereon at trackable loci and a record or data set including, for eachMS sample, a descriptor of the position of the trackable locus and anon-informative tracking identifier uniquely identifying the MS sample.

In an aspect, a descriptor of the position of the trackable locus of anMS sample includes a descriptor of the position of the trackable locusrelative to at least one physical characteristic of the MS substrate ora mounting frame on which the MS substrate is mounted, which may be afiducial mark.

In an aspect, a record or dataset includes, for an MS sample, adescriptor of the position of the center of the MS sample relative tothe at least one fiducial point, and the diameter of the MS sample.

In an aspect, a descriptor of the position of the trackable locus of anMS sample includes a descriptor of the position of the MS samplerelative to one or more other MS samples.

In an aspect, a MS record or dataset is written on a non-transitorymachine-readable medium in a file or data structure compatible withpositioning control software of an instrument selected from: an acousticapplication instrument, a moveable stage, a laboratory roboticsinstrument, or a MS instrument.

In an aspect, there is provided a substrate comprising a poroussemiconductor, which may further comprise a fluorous initiator, such as,for example, a fluorous siloxane, adsorbed to the semiconductor, and/ora photoactive compound containing a fluorous group, such as, forexample, a flourous group selected from perfluoro-1-butanesulfonate,N-hydroxynaphthalimide triflate and5,10,15,20-tetrakis(pentafluorophenyl) porphyrin, adsorbed to thesemiconductor.

In an aspect, there is provided a method of detecting an analyte in asample by desorption ionization mass spectrometry, which may include:(1) depositing a sample having a volume in the picoliter to nanoliterrange on a substrate, which may be, for example, by non-contactdeposition, such as, for example, acoustic deposition; (2) deliveringradiation to the sample to cause desorption and ionization of thesample; and (3) detecting the mass-to-charge ratio of the ionizedanalyte.

In an aspect, there is provided a method of detecting an analyte in asample by desorption ionization mass spectrometry, which may include:(1) depositing a plurality of matrix-free samples of differingcomposition, each having a volume in the picoliter to nanoliter range ona matrix-free substrate, which may be, for example, by non-contactdeposition, such as, for example, acoustic deposition; (2) deliveringradiation to each of the samples to cause desorption and ionization ofthe samples; and (3) detecting the mass-to-charge ratio of the ionizedanalyte from each sample.

For clarity and to ensure completeness, certain of the aspects and/orembodiments disclosed herein may be overlapping in scope, describedrepetitively, or represent recitals of the same or equivalent elementsor combinations expressed in alternative language. It will be apparentthat the choice of particular phraseology and/or of particular aspectsor elements to assert as claims involves many complex technical andlegal considerations, and no inference should be drawn that alternativedescriptions of a particular element or combination in this writtendescription necessarily encompass different subject matter.

It is intended that this specification be interpreted in accordance withthe normal principles of English grammar and that words and phrases begiven their ordinary English meaning as understood by persons of skillin the pertinent arts except as otherwise explicitly stated. If a word,term, or phrase is intended to be further characterized, specified, ornarrowed in some way, then additional adjectives, modifiers, ordescriptive text have been included in accordance with the normalprinciples of English grammar. It is intended that the meanings ofwords, terms, or phrases should not be modified or characterized in amanner differing from their ordinary English meaning as understood bypersons of skill in the relevant arts except on the basis of adjectives,modifiers, or descriptive text that is explicitly present.

Except as otherwise explicitly stated, terms used in this specification,including terms used in the claims and drawings, are intended as “open”terms. That is, for example, the words “including” and “comprising”should be interpreted to mean “including but not limited to,” the word“having” should be interpreted to mean “having at least,” the word“includes” should be interpreted to mean “includes but is not limitedto,” the phrases “for example” or “including by way of example” shouldbe interpreted as signifying that the example(s) given arenon-exhaustive and other examples could be given, and other similarwords and phrases should be given similar non-exclusive meanings.

As used herein, the term “about” or “approximately” means within 20%,preferably within 10%, and more preferably within 5% (or 1% or less) ofa given value or range. Various standard abbreviations and acronyms asdefined in J. Org. Chem. 2007 72(1):23A-24A are used herein. Otherabbreviations and acronyms used herein include: LDI: laser-desorptionionization.

In the written description and appended claims, the indefinite articles“a” and/or “an” are intended to mean “at least one” or “one or more”except where expressly stated otherwise or where the enabling disclosurerequires otherwise. The word “or” as used herein is intended to mean“and/or”, except where it is expressly accompanied by the word “either”,as in “either A or B”. Applicants are aware of the provisions of 35U.S.C. § 112, ¶6. The use of the words “function,” “means” or “step” inthe written description, drawings, or claims herein is not intended toinvoke the provisions of 35 U.S.C. § 112, ¶6, to define the invention.To the contrary, if the provisions of 35 U.S.C. § 112, ¶6 are sought tobe invoked, the claims will expressly include one of the exact phrases“means for performing the function of” or “step for performing thefunction of”. Moreover, even if the provisions of 35 U.S.C. § 112, ¶6are explicitly invoked to define a claimed invention, it is intendedthat the claims not be limited only to the specific structure, materialor acts that are described in the preferred embodiments, but inaddition, extend to any and all structures, materials or acts thatperform the claimed function as described in alternative embodiments orforms of the invention, or that are well known present orlater-developed equivalent structures, material or acts for performingthe claimed function.

Any of the methods of the present disclosure may be implemented in wholeor part in hardware, software, or both, or by a computer program, andmay be carried out using any of the disclosed devices or apparatusaccording to any aspect or embodiment of the present invention, or inany other operable manner.

In the foregoing description, various details, specific aspects,embodiments, and examples have been described in order to illustrate andexplain the subject matter, to provide a thorough understanding of thevarious aspects, to enable persons skilled in the pertinent arts topractice the described subject matter, and to disclose the best mode ofdoing so known to applicants. These details, specific aspects,embodiments, and examples are not intended to be limiting; rather, itwill be apparent to persons of skill in the relevant arts that, basedupon the teachings herein, various changes, substitutions,modifications, rearrangements, may be made and various aspects,components, or steps may be omitted or added, without departing from thesubject matter described herein and its broader aspects. Except asotherwise expressly stated or where aspects or features are inherentlymutually exclusive, aspects and features of any embodiment describedherein may be combined with aspects and features of any one or moreother embodiments. The appended claims are intended to encompass withintheir scope any and all changes, substitutions, modifications,rearrangements, combinations of aspects or features, additions, andomissions that are within the spirit and scope of the subject matter asdescribed herein and/or within the knowledge of a person of skill in theart. The scope of the invention is defined by the claims, and is notlimited by or to the particular embodiments or aspects chosen fordetailed exposition in the foregoing description, but rather extends toall embodiments or aspects as defined by the claims, as well as anyequivalents of such embodiments or aspects, whether currently known ordeveloped in the future.

The practice of various of the systems and methods disclosed hereinrelies on various conventional techniques in mass spectrometry andrelated fields such as are within the skill of the art. These techniquesare described in the references cited herein and are fully explained inthe literature. See, e.g., Siuzdak, Mass Spectrometry for Biotechnology(1996) Elsevier Science, USA; Dass, Fundamentals of Contemporary MassSpectrometry (2007) Wiley Interscience, Hoboken, N.J. So as to reducethe complexity and length of the detailed description, and to providebackground in certain areas of technology, the foregoing references aswell as each of the materials identified in the “REFERENCES” sectionbelow are expressly incorporated by reference. Applicants believe thatthe subject matter incorporated is “non-essential” in accordance with 37CFR 1.57, because it is referred to for purposes of indicating thebackground of the invention or illustrating the state of the art.However, if the Examiner concludes that any of the incorporated materialconstitutes “essential material” within the meaning of 37 CFR1.57(c)(1)-(3), applicants will amend the specification to expresslyrecite the essential material that is incorporated by reference asallowed by the applicable rules.

REFERENCES

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We claim:
 1. A method of providing a mass spectrometric (MS) analysisservice, comprising: receiving from a customer a pre-loaded MS array anda tracking record, wherein the MS array comprises a plurality ofnon-identical MS samples applied to an MS substrate and the trackingrecord associates each MS sample with a tracking identifier; performingMS analysis on each of the plurality of MS samples to produce MSanalysis data; associating MS analysis data corresponding to each MSsample with the tracking identifier of the MS sample in an MS datarecord; and providing access to the MS data record at the customer'sdirection; wherein the pre-loaded MS array comprises a plurality ofnon-identical MS samples applied to the MS substrate at a first locationand the MS analysis is performed at a second location offsite from thefirst location and the MS analysis comprises Laser Desorption/Ionization(LDI) of the samples directly from the MS array; wherein the trackingidentifier is a blinded tracking identifier and the tracking record doesnot reveal the chemical structure of the MS sample.
 2. The method ofclaim 1, wherein the tracking record associates a trackable locus ofeach MS sample with the tracking identifier of the MS sample.
 3. Themethod of claim 1, wherein the pre-loaded MS array comprises blinded MSsamples.
 4. The method of claim 1, wherein the pre-loaded MS array isreceived from the customer in a condition operable for performing MSanalysis of the samples in situ on the MS array without further samplepreparation.
 5. The method of claim 1, wherein the pre-loaded MS arrayis received from the customer in a condition operable for performinglaser desorption ionization (LDI) of the samples in situ on the MS arraywithout further sample preparation.
 6. The method of claim 1, whereinreceiving a pre-loaded MS array from a customer comprises receiving apre-loaded MS array dispatched by the customer for transport via acarrier.
 7. The method of claim 1, wherein providing access to the MSdata record at the customer's direction comprises incorporating the MSdata record in a data store and providing access to the data store to orat the direction of the customer.
 8. The method of claim 1, whereinproviding access to the MS data record at the customer's directioncomprises providing access to the MS data record over a communicationsnetwork.
 9. The method of claim 1, wherein providing access to the MSdata record at the customer's direction comprises providing the MS datarecord on a machine readable medium.
 10. The method of claim 1, whereinthe customer does not reveal the composition of the MS samples to theprovider of the MS analysis service.
 11. A data processing system forproviding an MS analysis service by an analysis provider, comprising: aplurality of MS sample data records obtained from a customer, eachcomprising a tracking identifier and a sample locator specifying thelocus of an MS sample on a pre-loaded MS array provided by the customerto the analysis provider and comprising a plurality of non-identical MSsamples disposed on an MS substrate; and a plurality of MS analysis datarecords each adapted and configured to store a tracking identifier andat least one MS characteristic of an MS sample with which the trackingidentifier is associated, wherein the at least one MS characteristic isdetermined by MS analysis performed on the MS sample by the analysisprovider and the MS analysis comprises Laser Desorption/Ionization (LDI)of the MS sample directly from the MS array: and a data store in whichthe plurality of MS analysis data records are stored and from which eachMS analysis data record is retrievable by a query comprising itstracking identifier; wherein the tracking identifier is a blindedtracking identifier and the tracking record does not reveal the chemicalstructure of the MS sample.
 12. The data processing system of claim 11,wherein each MS analysis data record is retrievable by or at thedirection of a customer entitled thereto.
 13. The data processing systemof claim 11, wherein the plurality of MS analysis data records isretrievable by or at the direction of a customer entitled thereto in aformat compatible for merging with a data store of the customer.
 14. Thedata processing system of claim 11, wherein each MS analysis data recorddoes not reveal chemical or physical characteristics of the MS sampleother than one or more characteristics determined by MS analysis of theMS sample.
 15. A method of using the data processing system of claim 11,comprising: receiving from a customer a query comprising the trackingidentifier of an MS sample; verifying that the customer is entitled toaccess an MS analysis data record that comprises the trackingidentifier; retrieving from the data store an MS analysis data recordthat comprises the tracking identifier; and delivering to the customerinformation contained in the MS analysis data record.
 16. Anon-transitory computer-readable storage medium containing instructionsfor execution by a processor of a computer system for querying a datastore of the data processing system of claim 11 with a query comprisinga tracking identifier and retrieving from the data store an MS analysisdata record comprising the tracking identifier.
 17. The data processingsystem of claim 11, wherein the data store comprises a data storeselected from: a relational database, an object-oriented database ordata structure, a no-SQL database, a cloud-based data storage utility,an XML or SGML-based data structure, a JSON data structure, a flat file;or a spreadsheet file.
 18. A non-transitory machine-readable mediumhaving data written thereon comprising a plurality of MS analysis datarecords, each MS analysis data record comprising a tracking identifierand an MS characteristic determined by offsite MS analysis of an MSsample with which the tracking identifier is associated, wherein the MSanalysis comprises Laser Desorption/Ionization (LDI) of the MS sampledirectly from a pre-loaded MS array received from a customer; whereinthe tracking identifier is a blinded tracking identifier and thetracking record does not reveal the chemical structure of the MS sample.19. The non-transitory machine-readable medium of claim 18 wherein thedata is written thereon in a format operable for merging with a datastore of a customer entitled to the results of an offsite MS analysis.